Interleukin-18 variants and methods of use

ABSTRACT

The present disclosure provides compositions and methods comprising stabilized IL-18 polypeptides for use in therapeutic and non-therapeutic applications. In some cases, the stabilized IL-18 proteins provide IL-18 signaling activity even in the presence of an inhibitory molecule such as IL-18BP. Also provide are methods of administration and methods for making active polypeptides.

CROSS-REFERENCE

This application is a continuation of International Application No.PCT/US2021/057741, filed on Nov. 2, 2021, which claims benefit of U.S.Provisional Application No. 63/108,794, filed on Nov. 2, 2020, each ofwhich is incorporated herein by reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in XML format and is hereby incorporated byreference in its entirety. Said XML copy, created on Nov. 14, 2022, isnamed 62106-703_301_SL.xml and is 228,044 bytes in size.

BACKGROUND

Interleukin 18 (IL-18) is a pro-inflammatory cytokine that can stimulateT cells, NK cells, and myeloid cells. IL-18 has been proposed as animmunotherapeutic agent for the treatment of cancer, given its abilityto stimulate anti-tumor immune cells. However, the clinical efficacy ofIL-18 has been limited.

Thus, there is a need for compositions and methods that provideeffective IL-18 signaling activity to treat and prevent cancer and otherdiseases and disorders. The present disclosure addresses these needs.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

SUMMARY OF THE INVENTION

In one aspect, the present disclosure provides a method of promotinginterleukin-18 (IL-18) signaling activity, the method comprisingadministering a modified IL-18 polypeptide to a subject in need thereof,the modified IL-18 polypeptide comprising: (i) an amino acid sequencehaving 85% or more sequence identity with the IL-18 variant aminosequence set forth in any one of SEQ ID NO: 89-91; and (ii) mutations atamino acid positions Cysteine-38 and Cysteine-68 relative to wild-type(WT) IL-18 as set forth in SEQ ID NO: 30, thereby promoting IL-18signaling activity.

In some embodiments, the subject is a human. In some embodiments, themutation at position Cysteine-68 is a substitution of Cysteine-68 toserine. In some embodiments, the mutation at position Cysteine-68 is asubstitution of Cysteine-68 to glycine. In some embodiments, themutation at position Cysteine-68 is a substitution of Cysteine-68 toalanine. In some embodiments, the mutation at position Cysteine-68 is asubstitution of Cysteine-68 to aspartic acid. In some embodiments, themutation at position Cysteine-68 is a substitution of Cysteine-68 toasparagine.

In some embodiments, the IL-18 polypeptide has one or more substitutionsselected from the group consisting of: (1) Tyrosine-1 to histidine, orTyrosine-1 to arginine, (2) Leucine-5 to histidine, Leucine-5 toisoleucine, or Leucine-5 to tyrosine, (3) Lysine-8 to glutamine, orLysine-8 to arginine, (4) Methionine-51 to threonine, Methionine-51 tolysine, Methionine-51 to aspartic acid, Methionine-51 to asparagine,Methionine-51 to glutamic acid, or Methionine-51 to arginine, (5)Lysine-53 to arginine, Lysine 53-glycine, Lysine-53 to serine, orLysine-53 to threonine, (6) Serine-55 to lysine, or Serine-55 toarginine, (7) Glutamine-56 to glutamic acid, Glutamine-56 to alanine,Glutamine-56 to arginine, Glutamine-56 to valine, Glutamine-56 toglutamic acid, Glutamine-56 to glycine, Glutamine-56 to lysine, orGlutamine-56 to leucine, (8) Proline-57 to leucine, Proline-57 toglycine, Proline-57 to alanine, or Proline-57 to lysine, (9) Glycine-59to threonine, or Glycine-59 to alanine, (10) Methionine-60 to lysine,Methionine-60 to glutamine, Methionine-60 to arginine, or Methionine-60to leucine, (11) Glutamic acid-77 to aspartic Acid, (12) Glycine-103 toglutamic acid, Glycine-103 to lysine, Glycine-103 to proline,Glycine-103 to alanine, or Glycine-103 to arginine, (13) Serine-105 toarginine, Serine-105 to aspartic acid, Serine-105 to asparagine, orSerine-105 to alanine, (14) Aspartic acid-110 to histidine, Asparticacid-110 to lysine, Aspartic acid-110 to asparagine, Aspartic acid-110to glutamine, Aspartic acid-110 to glutamic acid, Aspartic acid-110 toserine, or Aspartic acid-110 to glycine, (15) Asparagine-111 tohistidine, Asparagine-111 to tyrosine, Asparagine-111 to aspartic acid,Asparagine-111 to arginine, Asparagine-111 to serine, or Asparagine-111to glycine, (16) Methionine-113 to valine, Methionine-113 to arginine,Methionine-113 to threonine, or Methionine-113 to lysine, (17)Valine-153 to isoleucine, Valine-153 to threonine, or Valine-153 toalanine, and (18) Asparagine-155 to lysine, or Asparagine-155 tohistidine, relative to SEQ ID NO: 30.

In some embodiments, the IL-18 polypeptide is a decoy resistant (DR)IL-18 variant or a decoy-to-the-decoy (D2D) IL-18 variant.

In some embodiments, the DR IL-18 variant comprises mutations in atleast two of the following positions: Tyrosine-1, Leucine-5, Lysine-8,Methionine-51, Lysine-53, Serine-55, Glutamine-56, Proline-57,Glycine-59, Methionine-60, Glutamic acid-77, Glutamine-103, Serine-105,Aspartic acid-110, Asparagine-111, Methionine-113, Valine-153, andAsparagine-155, relative to SEQ ID NO: 30.

In some embodiments, the DR IL-18 variant comprises mutations in atleast two of the following positions: Methionine-51, Lysine-53,Serine-55, Glutamine-56, Proline-57, Methionine-60, Glutamine-103,Serine-105, Aspartic acid-110, Asparagine-111, and Methionine-113,relative WT IL-18 as set forth in SEQ ID NO: 30.

In some embodiments, the DR IL-18 variant comprises mutations atpositions Methionine-51, Lysine-53, Glutamine-56, Aspartic acid-110, andAsparagine-111, relative to WT IL-18 as set forth in SEQ ID NO: 30. Insome embodiments, the DR IL-18 variant further comprises mutations atpositions Proline-57 and Methionine-60, relative to WT IL-18 as setforth in SEQ ID NO: 30. In some embodiments, the DR IL-18 variantfurther comprises a mutation at position Serine-105, relative to WTIL-18 as set forth in SEQ ID NO: 30.

In some embodiments, the DR IL-18 variant has at least fivesubstitutions selected from the group consisting of: (1) Methionine-51to threonine, Methionine-51 to lysine, Methionine-51 to aspartic acid,Methionine-51 to asparagine, Methionine-51 to glutamic acid, orMethionine-51 to arginine, (2) Lysine-53 to arginine, Lysine-53 toglycine, Lysine-53 to serine, or Lysine-53 to threonine, (3)Glutamine-56 to glutamic acid, Glutamine-56 to alanine, Glutamine-56 toarginine, Glutamine-56 to valine, Glutamine-56 to glycine, Glutamine-56to lysine, or Glutamine-56 to leucine, (4) Proline-57 to leucine,Proline-57 to glycine, Proline-P57 to alanine, or Proline-57 to lysine,(5) Methionine-60 to lysine, Methionine-60 glutamine, Methionine-60 toarginine, or Methionine-60 to leucine, (6) Glutamine-103 to glutamicacid, Glutamine-103 to lysine, Glutamine-103 to proline, Glutamine-103to alanine, or Glutamine-103 to arginine, (7) Serine-105 to arginine,Serine-105 to aspartic acid, Serine-105 to lysine, Serine-105 tohistidine, or Serine-105 to alanine, (8) Aspartic acid-110 to histidine,Aspartic acid-110 to lysine, Aspartic acid-110 to asparagine, Asparticacid-110 to glutamine, Aspartic acid-110 to glutamic acid, Asparticacid-110 to serine, Aspartic acid-110 to glycine, (9) Asparagine-111 tohistidine, Asparagine-111 to tyrosine Asparagine-111 to aspartic acid,Asparagine-111 to arginine, Asparagine-111 to serine, or Asparagine-111to glycine, and (10) Methionine-113 to valine, Methionine-113 toarginine, Methionine-113 to threonine, or Methionine-113 to lysine,relative to SEQ ID NO: 30.

In some embodiments, the DR IL-18 variant has at least fivesubstitutions selected from the group consisting of: (1) Methionine-51to glutamic acid, Methionine-51 to arginine, Methionine-51 to lysine,Methionine-51 to threonine, Methionine-51 to aspartic acid, orMethionine-51 to asparagine; (2) Lysine-53 to glycine, Lysine-53 toserine, Lysine-53 to threonine, or Lysine-53 to arginine; (3)Glutamine-56 to glycine, Glutamine-56 to arginine, Glutamine-56 toleucine, Glutamine-56 to glutamic acid, Glutamine-56 to alanine, Q56V,or Glutamine-56 to lysine; (4) Aspartic acid-110 to serine, Asparticacid-110 to asparagine, Aspartic acid-110 to glycine, Aspartic acid-110to lysine, Aspartic acid-110 to histidine, Aspartic acid-110 toglutamine, or Aspartic acid-110 to glutamic acid; (5) Asparagine-111 toglycine, Asparagine-111 to arginine, Asparagine-111 to serine,Asparagine-111 to aspartic acid, Asparagine-111 to histidine, orAsparagine-111 to tyrosine; (6) Proline-57 to alanine, Proline-57 toleucine, Proline-57 to glycine, or Proline-57 lysine; and (7)Methionine-60 to leucine, Methionine-60 to arginine, Methionine-60 tolysine, or Methionine-60 to glutamine, relative to SEQ ID NO: 30. Insome embodiments, the DR IL-18 variant further comprises thesubstitution Serine-105 to aspartic acid, Serine-105 to alanine,Serine-105 to asparagine, Serine-105 to arginine, Serine-105 to asparticacid, or Serine-105 to lysine relative to SEQ ID NO: 30.

In some embodiments, the DR IL-18 variant specifically binds to IL-18receptor (IL-18R) but exhibits reduced binding to IL-18 binding protein(IL-18BP) compared to the WT IL-18. In some embodiments, the DR IL-18variant comprises substitutions of Methionine-50 to lysine, Lysine-53 toserine, Glutamine-56 to leucine, Aspartic acid-110 to serine, andAsparagine-111 to arginine, relative to SEQ ID NO: 30. In someembodiments, the DR IL-18 variant further comprises substitutions ofProline-57 to alanine, Methionine-60 to leucine, and Serine-105 toaspartic acid.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of embodiments of the invention willbe better understood when read in conjunction with the appendeddrawings. It should be understood that the invention is not limited tothe precise arrangements and instrumentalities of the embodiments shownin the drawings.

FIG. 1A and FIG. 1B depict results from example experiments,demonstrating the IL-18 pathway is a target for tumor immunotherapy.FIG. 1A provides a chart showing that the IL-18 pathway (including IL-18and its receptor subunits) is upregulated in both activated anddysfunctional tumor T cell programs, as seen in RNAseq expressionanalysis for cytokines and receptors in CD8+ TILs. Genes are assigned“activation” and “dysfunction” scores in comparison to naïve T cells, asseen in the chart. Data points marked with a diamond-shape and blacktext indicate IL-18 cytokine, IL-18R1 (Ra), and IL-18RAP (Rβ). Data areadapted from Singer et al. (Singer, M. et al., 2016, Cell,166:1500-1511, e1509). FIG. 1B provides the results of infection withLCMV (left; CD4) or VSV-OVA (right; CD8). The IL-18 receptor subunitsIL-18Rα and IL-18Rβ are part of a gene expression program associatedwith chronic antigen exposure, as seen after infection with LCMV (left;CD4) or VSV-OVA (right; CD8). Data are from the ImmGen database.

FIG. 2A through FIG. 2C depict results from example experiments,demonstrating IL-18BP has features of a “soluble immune checkpoint”.FIG. 2A shows how the IL-18BP mediates Interferon-γ (IFN-γ) drivennegative feedback of IL-18, reminiscent of the immune checkpoint PD-L1.A schematic of the IL-18/IFN-γ/IL-18BP feedback loop is depicted. Blackarrows indicate stimulation, dark gray circuits indicate inhibition.FIG. 2B shows that IL-18BP is upregulated in gastric and breast cancerusing data from the TCGA and Oncomine databases. FIG. 2C demonstratesthat PD-1 and IL-18BP expression is strongly correlated in bulk breastand gastric cancer samples (from TCGA database). R=0.78 and 0.65,respectively.

FIG. 3A through FIG. 3C depict results from example experiments,demonstrating engineering human IL-18 variants for independence toIL-18BP using yeast display. FIG. 3A provides a structure-guidedlibrary, which was designed to randomize residues on the IL-18:IL-18BPinterface and introduced into a yeast-display system. Yeast clones wereselected using magnetic and fluorescence cell sorting for binding toIL-18Rα and counter-selected against IL-18BP. FIG. 3B provides a summaryof directed evolution to generate IL-18BP resistant IL-18 variants.Positive selection conditions are: hIL18Rα SA-beads and hIL18Ra.Counterselection conditions are: SA-alone, hIL-18BP, and nIL-18BPtetramer. FIG. 3C provides flow cytometric analysis of yeast-displayedWT IL-18 (left) or variants after directed evolution (right). Y-axesshow IL-18BP binding, x-axes show IL-18Rα binding. After 5 rounds ofdirected evolution, the remaining clones greatly preferred IL-18Rα toIL-18BP.

FIG. 4 depicts results from example experiments, demonstrating a summaryof the sequences of decoy-resistant human IL-18 (“DR-IL-18”, also called“DR-18”) variants. The position of each mutated position and thecorresponding residue in the mature form of wild-type human IL-18 isindicated at the top of the table. SEQ ID NO: 38 through SEQ ID NO: 58represent sequences obtained after selection with directed evolution.SEQ ID NO: 34-SEQ ID NO: 37 are consensus sequences derived from theselected sequences. Shaded residues represent the five most conservedmutations observed. The sequence for the top row (“WT hIL-18”) is setforth as SEQ ID NO: 30.

FIG. 5A and FIG. 5B depict results from example experiments,demonstrating biophysical characterization of human DR-IL-18 variants.FIG. 5A provides binding isotherms on yeast-displayed DR-hIL-18variants. As shown, yeast-displayed DR-IL-18 variants SEQ ID NO: 34-37and SEQ ID NO: 39 are capable of binding hIL-18Rα with comparablebinding isotherms as WT human IL-18 (left). By contrast, very littlebinding is observed with the same variants and hIL-18BP (right). FIG. 5Bshows representative surface plasmon resonance sensor grams betweenimmobilized biotinylated hIL-18BP and the DR-IL-18 variants. RecombinanthIL-18 (left) binds IL-18BP with exquisitely high affinity, KD=2.0 pM,whereas SEQ ID NO: 34 (right) shows greatly attenuated binding, with amuch faster off-rate and KD=15.2 nM. This data is summarized in Table 6and Table 7.

FIG. 6A and FIG. 6B depict results from example experiments,demonstrating human DR-IL-18 variants are not inhibited by IL-18BP. FIG.6A provides a chart showing that recombinant IL-18BP inhibitsbiotinylated IL-18Rα from binding yeast-displayed WT IL-18, but does notaffect the DR-IL-18 variants SEQ ID NO: 34-37 and SEQ ID NO: 39 (shownin chart on left). By contrast, IL-18BP effectively neutralizes theIL-18 E42A, K89A and E42A/K89A previously described (Kim et al., 2001,Proc. Natl. Acad. Sci., 98(6):3304-3309) (shown in chart on right) [E42and K89 of Kim et al. are E6 and K53 of SEQ ID NO: 30, respectively].Biotinylated IL-18Rα was kept at a fixed concentration of 100 nM for allsamples. FIG. 6B provides results of HEK Blue IL-18 signaling assay,showing that WT IL-18 and SEQ ID NO: 34, SEQ ID NO: 36, and SEQ ID NO:37 stimulate IL-18 HEK-Blue reporter cells with comparable potency andefficacy (left). Wild-type IL-18 is highly sensitive to application ofrecombinant IL-18BP in this assay (IC50=3 nM), whereas SEQ ID NO: 34 andSEQ ID NO: 36 are not inhibited by recombinant IL-18BP, even at IL-18BPconcentrations of 1 μM (Right). hIL-18 was kept at a fixed concentrationof 5 nM and SEQ ID NO: 34 and SEQ ID NO: 36 at 2.5 nM.

FIG. 7A through FIG. 7C depict results from example experimentsdemonstrating engineering additional human IL-18 variants forindependence to IL-18BP (version 2 variants) using yeast display. FIG.7A provides a summary of the positions in human IL-18 randomized in theversion 2.0 library. Degenerate codons and the set of encoded aminoacids are given for each position. FIG. 7B provides a summary ofdirected evolution to generate version 2.0 IL-18BP resistant IL-18variants. Positive selection conditions are: hIL18Rα SA-beads andhIL18Rα Counterselection conditions are: SA alone, hIL-18BP, andhIL-18BP tetramer. FIG. 7C provides a flow cytometric analysis ofprogress in creating version 2.0 DR-IL-18 variants. Yeast obtained afterrounds 1, 4, and 6 were stained simultaneously with 250 nM IL-18BPstreptavidin-PE tetramers or 100 nM IL-18Rα directly labeled withAlexaFluor647. Y-axes show IL-18BP binding, x-axes show IL-18Rα binding.After 6 rounds of directed evolution, the remaining clones greatlypreferred IL-18Rα to IL-18BP.

FIG. 8 depicts results from example experiments, demonstrating a summaryof the sequences of version 2.0 decoy-resistant human IL-18 (DR-IL-18)variants. The position of each mutated position and the correspondingresidue in the mature form of wild-type human IL-18 is indicated at thetop of the table. Shaded rows indicate recurrent sequence variantsobtained in both round 5 and round 6. The sequence for the top row (“WThIL-18”) is set forth as SEQ ID NO: 30.

FIGS. 9A-9D depict results from example experiments, demonstratingbiophysical characterization of version 2.0 human DR-IL-18 variants.FIG. 9A shows that yeast-displayed version 2.0 DR-IL-18 variants arecapable of binding hIL-18Rα with comparable binding isotherms as WThuman IL-18. FIG. 9B shows, by contrast, that very little binding isobserved with the same variants and hIL-18BP. FIG. 9C provides thermalstability of the version 2.0 DR-IL-18 variants, as was assessed byheating the yeast-displayed variants across a range of temperatures for15 minutes, followed by staining with hIL-18Ra. The version 2.0 DR-IL-18variants were more thermostable than WT IL-18 (Tm=47.6 C) and thefirst-generation consensus sequences (Tm=50.9 and 40.2 for SEQ ID NO: 24and SEQ ID NO: 35, respectively). FIG. 9D provides a summary of thereceptor binding properties and thermal stability of thesecond-generation DR-IL-18 variants. NBD=no binding detected. N.D.=valuenot determined.

FIG. 10A through FIG. 10C depict results from example experiments,demonstrating engineering murine IL-18 variants for independence toIL-18BP using yeast display. FIG. 10A provides a summary of directedevolution to generate IL-18BP resistant murine IL-18 variants. Positiveselection conditions are: mIL18Ra SA-beads and mIL18Ra. Counterselectionconditions are: mIL-18BP and mIL-18BP tetramer. FIG. 10B shows resultsof flow cytometric analysis of yeast-displayed murine IL-18 variantsafter 5 rounds of directed evolution. Y-axes show IL-18BP binding,x-axes show IL-18Rα binding. FIG. 10C provides a summary of thesequences of decoy-resistant murine IL-18 (DR-IL-18) variants. Thesequence for the top row (“mIL-18”) is set forth as SEQ ID NO: 31. Theposition of each mutated position and the corresponding residue in themature form of wild-type murine IL-18 is indicated at the top of thetable. SEQ ID NO: 62 through SEQ ID NO: 72 represent sequences obtainedafter selection with directed evolution. SEQ ID NO: 60 and SEQ ID NO: 61are consensus sequences derived from the selected sequences. Shadedresidues represent the five most conserved mutations observed.

FIG. 11A and FIG. 11B depict results from example experiments,demonstrating biophysical characterization of murine DR-IL-18 variants.FIG. 11A shows that yeast-displayed DR-IL-18 variants SEQ ID NO: 70, SEQID NO: 67, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 60, and SEQ ID NO:61 are capable of binding mIL-18Rα with comparable binding isotherms asWT murine IL-18 (left). By contrast, very little binding is observedwith the same variants and mIL-18BP (right). FIG. 11B providesmeasurement of IL-18BP binding using representative surface plasmonresonance sensor grams between immobilized biotinylated mIL-18BP and themurine DR-IL-18 variants. Recombinant mIL-18 (left) binds mIL-18BP withhigh affinity, KD=0.8 pM, whereas SEQ ID NO: 61 (right) shows greatlydecreased binding with a KD value greater than 10 NM. This data issummarized in Tables 8 and 9.

FIG. 12A through FIG. 12D depict results from example experiments,demonstrating pharmacodynamics of DR-IL-18 administered to mice. FIG.12A is a schematic of study design. Mice were administered vehicle(PBS), mIL-18 (1 mg/kg), or the DR-IL-18 variant SEQ ID NO: 61 (1 mg/kg)once daily for seven total doses (depicted as syringes). Blood sampleswere taken five hours post-injection two days prior to the experiment,and on days 0, 3, and 6. FIG. 12B shows peripheral blood cell counts forCD4, CD8, NK cells, and monocytes at day 0, day 3, and day 6. Both IL-18and SEQ ID NO: 61 expanded NK cells and monocytes to a similar degree byday 3. For each time point (day), left bar is PBS, middle bar is IL-18,and right bar is SEQ ID NO: 61. FIG. 12C shows the CD69 expression onperipheral CD4, CD8, and NK cells. SEQ ID NO: 61, but not IL-18stimulated CD69 expression on CD4 and CD8 cells. Both IL-18 and SEQ IDNO: 61 increased CD69 on NK cells, but SEQ ID NO: 61 treatment causedsustained CD69 expression evident at day 6, in comparison to IL-18,which reverted to baseline CD69 levels. For each time point (day), leftbar is PBS, middle bar is IL-18, and right bar is SEQ ID NI: 61. FIG.12D provides Serum cytokine levels for interferon-g (IFN-g), MIP-1b, andG-CSF. SEQ ID NO: 61 treatment yielded higher levels of IFN-g, MIP-1b,and G-CSF than mIL-18 treatment.

FIG. 13 depicts results from example experiments, demonstrating DR-IL-18treatment decreases body fat composition in mice. Body fat and lean masscomposition were measured in mice treated with 0.01, 0.1, or 1 mg/kg ofthe DR-IL-18 variant SEQ ID NO: 61 or 1 mg/kg WT mIL-18 every threedays. SEQ ID NO: 61 treatment produced a significant decrease in bodyfat as a total percentage of body mass (top panel). This was manifestedby decreases or stable fat mass (left panel), with concordant increasesin lean mass (right panel). Vehicle treated and mIL-18 treated miceshowed increases in body fat mass and stable lean mass over the sametreatment period.

FIG. 14A through FIG. 14B depict results from example experiments,demonstrating DR-IL-18 is an effective immunotherapeutic in a melanomamodel. FIG. 14A provides tumor growth spider plots for mice bearingYummer1.7 melanoma tumors treated with saline (control), WT IL-18 (0.32mg/kg), the DR-IL-18 variant SEQ ID NO: 61 (0.32 mg/kg), anti-PD1 (8mg/kg), IL-18+anti-PD1, or SEQ ID NO: 61+anti-PD-1 twice per week. FIG.14B provides survival curves from the same groups as in FIG. 14A. Asshown in FIG. 14B, SEQ ID NO: 61 was effective as a monotherapy andsynergized in combination with anti-PD1 in this model.

FIG. 15A and FIG. 15B depict results from example experiments thatdemonstrate that the effectiveness of DR-IL-18 in the melanoma model ofFIG. 14 is dependent on CD4 and CD8 lymphocytes and interferon gamma.FIG. 15A provides tumor growth spider plots for mice bearing Yummer1.7melanoma tumors treated with saline (control), or the DR-IL-18 variantSEQ ID NO: 61 (0.32 mg/kg) alone, or in combination with depletingantibodies against CD8, CD4, interferon gamma, or NK1.1. FIG. 15Bprovides survival curves from the same groups as in FIG. 15A.

FIG. 16 depicts results from example experiments, demonstratingdose-dependent efficacy of DR-IL-18 in the MC38 tumor model. Tumorgrowth spider plots from mice bearing MC38 colon cancer tumors treatedwith PBS (control), 1.0 mg/kg WT IL-18, 1.0 mg/kg SEQ ID NO: 61, 0.1mg/kg SEQ ID NO: 61, or 0.01 mg/kg SEQ ID NO: 61 every three days. WTIL-18 was not efficacious at 1 mg/kg, whereas SEQ ID NO: 61 showedpartial efficacy at 0.1 mg/kg and maximal efficacy at 1.0 mg/kg.

FIG. 17 depicts results from example experiments demonstrating theefficacy of DR-IL-18 alone in combination with the immune checkpointinhibitor anti-PD1 in the MC38 tumor model. Tumor growth spider plotsare shown from mice bearing MC38 colon cancer tumors treated with PBS(control), 0.32 mg/kg WT IL-18, 0.32 mg/kg of the DR-IL-18 variant SEQID NO: 61, 5 mg/kg anti-PD1, the combinations of anti-PD1 with WT IL-18,or the combination of anti-PD1 with SEQ ID NO: 61. All agents were dosedintraperitoneally twice per week for up to 6 total doses.

FIG. 18A and FIG. 18B depict results from example experiments thatinvestigate the anti-tumor mechanism of DR-IL-18 in mice bearing MC38tumors. FIG. 18A provides results of tumor immunophenotyping experimentsfrom mice treated twice weekly for four doses with saline, WT IL-18, orthe DR-IL-18 variant SEQ ID NO: 61. DR-IL-18 treatment resulted inincreased numbers of CD8 and NK cells per mg of tumor (upper left twopanels) and increased expression of activation markers granzyme B andKLRG1 on CD8 and NK cells (upper right two panels). DR-IL-18 treatmentdid not improve the CD8:Treg ratio compared to saline treatment, whereasWT IL-18 made the ratio less favorable. However, DR-IL-18 treatmentincreased the ratio of CD8 cells to inhibitory myeloid populationsincluding tumor associated macrophages (TAM), and monocytic andgranulocytic myeloid derived suppressor cells (mMDSCs and gMDSCs). FIG.18B provides Serum Luminex cytokine measurements from the same mice asFIG. 18A, taken 24 hours after the 4th treatment dose. DR-IL-18 shows adramatically altered secondary cytokine release profile from treatmentwith WT IL-18, notably increasing Interferon-gamma, IL-7, and IL-15levels by more than 100-fold.

FIG. 19A through FIG. 19C depicts results from example experimentsdemonstrating the capability of DR-IL-18 to effectively treat tumorsthat are refractory to immune checkpoint inhibitors through loss ofsurface MHC class I expression. FIG. 19A shows tumor growth spider plotsfrom mice bearing B2m-deficient Yummer1.7 tumors treated with saline,anti-PD1+anti-CTLA4, or the DR-IL-18 variant SEQ ID NO: 61. FIG. 19Bcontains a chart showing percent survival versus days post engraftment.DR-IL-18 demonstrated strong efficacy in terms of tumor growth andsurvival, curing 60% of treated mice in this model that is completelyresistant to even combination treatment with anti-CTLA4+anti-PD1. Thisefficacy is NK cell dependent since administration of DR-IL-18 withanti-NK1.1 (which depletes NK cells) abrogates the SEQ ID NO: 61treatment effect. FIG. 19C provides plots comparing Interferon-gammaproduction and Ki67 levels in tumors. NK cells isolated fromB2m-deficient Yummer1.7 are dysfunctional and show diminishedproliferation (Ki67 staining) and function (Interferon-gamma secretion).However, treatment with DR-IL-18 reverses this phenotype to enablerobust proliferation and cytokine secretion.

FIG. 20A through FIG. 20C depicts example experiments demonstratingengineering of human IL-18 variants as IL-18BP antagonists (or“decoys-to-the-decoy”, D2D) using yeast display. These variants bindIL-18BP but do not signal, thereby antagonizing the effect of IL-18BP onendogenous IL-18. FIG. 20A is a summary of the positions in human IL-18randomized in the D2D library. Degenerate codons and the set of encodedamino acids are given for each position. FIG. 20B provides a summary ofdirected evolution to generate D2D IL-18 variants that bind andneutralize IL-18BP, but do not signal through the IL-18R. The positiveselection condition is mIL18BP. Counter-selection conditions are:nIL18Rα, hIL18Rb, and mIL18Rα. (FIG. 20C) Flow cytometric analysis ofprogress in creating D2D hIL-18 variants. Yeast obtained after rounds1˜4 were stained with 1 nM of mouse IL-18BP (left panel), 1 nM humanIL-18BP (middle panel), or 1 μM IL18Ra plus 1 μM IL18Rβ. Selectedvariants show enhanced IL-18BP binding across rounds of selectionwithout increases in IL18Rα or IL18Rβ binding.

FIG. 21 depicts results from example experiments demonstrating a summaryof the sequences of D2D human IL-18 variants. The position of eachmutated position and the corresponding residue in the mature form ofwild-type human IL-18 is indicated at the top of the table. The sequencefor the top row (“WT hIL-18”) is set forth as SEQ ID NO: 30.

FIG. 22A through FIG. 22C depict results from example experimentsdemonstrating biophysical characterization of the humandecoy-to-the-decoy (D2D) IL-28 variants. FIG. 22A shows thatyeast-displayed D2D IL-8 variants SEQ ID NO: 120, SEQ ID NO: 101, SEQ IDNO: 94, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 123, SEQ ID NO: 124,and SEQ ID NO: 125 are capable of binding hIL-18RBP with comparablebinding isotherms as WT human IL-18. FIG. 22B shows that, by contrast,very little binding is observed with the same variants and hIL-18Ra.FIG. 22C provides a summary of the receptor binding properties of theD2D IL-18 variants. NBD=no binding detected.

FIG. 23 depicts results from example experiments demonstrating a summaryof the sequences of D2D murine IL-18 variants. The position of eachmutated position and the corresponding residue in the mature form ofwild-type murine IL-18 is indicated at the top of the table. Thesequence for the top row (“WT mIL-18”) is set forth as SEQ ID NO: 31.

FIG. 24 depicts results from biophysical affinity measurements(sensograms) of the second-generation DR-IL-18 variants for binding toIL-18Rα and IL-18BP using Surface Plasmon Resonance (SPR). Top row:representative sensograms of the indicated IL-18 variants (solubleanalytes) for hIL-18Rα (immobilized ligand). Bottom row: representativesensograms of the indicated IL-18 variants for human (hIL-18BP). The xaxis is time in seconds and the y axis is Response Units (RU). Thecurves are the observed data over time for different concentrations(2-fold dilutions starting at 1 nM), superimposed with curves of bestfit assuming a 1:1 langmuir binding model.

FIG. 25A and FIG. 25B depict data demonstrating efficacy of DR-IL-18 onthe CT26 colorectal tumor model. 250,000 CT26 cells were implantedsubcutaneously and treatment initiated at day 7 once tumors were −60 mm³on average. WT IL-18 and SEQ ID NO: 61 were dosed at 0.32 mg/kg twiceweekly for a total of 5 doses. Anti-PD1 was given at 10 mg/kg at thesame schedule. FIG. 25A is an overlay of spider plots showing tumorgrowth of saline (PBS) treated animals in black lines (circles), WTIL-18 in dark gray (squares), and DR-IL-18 (SEQ ID NO: 61) in gray(triangles). Only treatment with DR-IL-18, but not WT IL-18, resulted intumor growth inhibition and tumor clearance in a subset of animals. FIG.25B comprises survival curves for mice treated with anti-PD-1, WT IL-18,and DR-IL-18 (SEQ ID NO: 61). Numbers of complete responses areindicated in parentheses. DR-IL-18, but not WT IL-18 resulted inprolonged survival and tumor clearance in 40% of mice, an improvementover the checkpoint inhibitor anti-PD-1.

FIG. 26A and FIG. 26B depict data demonstrating efficacy of DR-IL-18 inthe 4T1 breast cancer model and B16-F10 melanoma model. FIG. 26A showstumor growth curves of 4T1 tumors engrafted into BALB/C mice aftertreatment with saline (PBS; black), WT IL-18 (dark gray), or theDR-IL-18 variant SEQ ID NO: 61 (gray). FIG. 26B shows tumor growthcurves of B16-F10 tumors engrafted into C57BL/6 mice after treatmentwith saline (PBS; black), WT IL-18/TA99 (dark gray), or the DR-IL-18variant SEQ ID NO: 61 (gray). In both models, only DR-IL-18, but not WTIL-18 resulted in tumor growth inhibition. Treatments were administeredafter tumors exceeded an average volume 50 mm³ as indicated by the boxesmarked with “t”.

FIG. 27A and FIG. 27B depict data that extend the data of FIG. 19Athrough 19C. Depicted is data demonstrating efficacy of DR-IL-18 in thetreatment of additional MHC class I deficient tumor models that areresistant to immune checkpoint inhibitors. FIG. 27A provides a chart oftumor volume versus days post implantation for mice implanted with B2mdeficient MC38 cells, which were prepared using CRISPR/Cas9 mediateddeletion as described for B2m deficient YUMMER cells. B2m−/− MC38 cellswere implanted subcutaneously and treatment initiated at day 7 oncetumors were −65 mm³ on average. SEQ ID NO: 61 was dosed at 0.32 mg/kgtwice weekly for 5 doses. Anti-PD1 and anti-CTLA4 were given at 8 mg/kgat the same schedule. FIG. 27B shows a chart of tumor volume versus dayspost implantation for mice implanted with RMA/S cells. RMA/S is avariant of the RMA lymphoma line that contains a spontaneous mutation inTapasin. The result is a defect in antigen loading and thereforedecreased MHC class I surface expression. It is congenic to C57BL/6 andrefractory to immune checkpoint inhibitors. Mice were implanted with1,000,000 RMA/S cells subcutaneously and treatment initiated at day 7.SEQ ID NO: 61 was dosed at 0.32 mg/kg twice weekly. Anti-PD1 was givenat 8 mg/kg at the same schedule. In both studies, only treatment withthe DR-18 variant SEQ ID NO: 61 exhibited anti-tumor efficacy in theform of tumor growth inhibition (B2m^(−/−) MC38) or tumor clearance(RMA/S).

FIG. 28 depicts data demonstrating efficacy of DR-IL-18 to enhanceanti-tumor antibody-dependent cell mediated cytotoxicity (ADCC). Ex vivocytotoxicity studies used CFSE labeled Raji (B cell lymphoma) cells andisolated human peripheral blood mononuclear cells (PBMCs). PBMCs andlabeled Raji cells were incubated together at an effector: target (E:T)ratio of 1:10 for 25 hours. The human DR-IL-18 variant hCS-1 (1 μM),rituximab (10 μg/mL), or the combination of both agents were applied tothe samples as indicated. Cytotoxicity was measured by flow cytometryand calculated as the fraction of CFSE cells that became DAPI positive.DR-18 stimulated significant tumor cell killing as a single agent andsignificantly enhanced the killing by the therapeutic antibodyrituximab. * p<0.05 by two-way ANOVA with Tukey's correction formultiple comparisons.

FIG. 29A and FIG. 29B depict data demonstrating anti-viral efficacy ofDR-18 variant SEQ ID NO: 61 for the treatment viral infections (e.g., inthis case in the treatment of systemic vaccinia virus infection). FIG.29A shows the experimental design scheme. C57BL/6 mice were infectedwith 106 PFU of Vaccinia virus (VACV) intraperitoneally (IP) andadministered 1 mg/kg WT mIL-18 or SEQ ID NO: 61 IP. Mice were sacrificedand viral titers were measured in the blood and ovaries by RT-PCR on day3 post-infection. FIG. 29B. demonstrates the quantification of VACVviral copies in ovaries and blood of treated mice at day 3 postinfection. Treatment with SEQ ID NO: 61 showed a significant reductionof viral titers, whereas WT IL-18 was not effective. * p<0.05, **p<0.01, *** p<0.001.

FIG. 30 depicts data demonstrating that the second-generation humanDR-IL-18 variants are active. (FIG. 30 ) WT IL-18 and human SEQ ID NO:89, SEQ ID NO: 90, SEQ ID NO: 91, and SEQ ID NO: 87 stimulate IL-18HEK-Blue reporter cells. Human SEQ ID NO: 89, SEQ ID NO: 90, and SEQ IDNO: 91 show enhanced potency compared to WT hIL-18, whereas SEQ ID NO:87 has equivalent potency as WT hIL-18. The data demonstrate, therefore,that all tested second generation human DR-IL-18 variants activelysignal through IL-18R.

FIG. 31 depicts IL-18 variants with mutations at various cysteinepositions that were prepared and then run on an SDS/PAGE gel (visualizedusing Spyro-Ruby stain). R=Reduced sample; N=Non-reduced sample;MW=Molecular weight. The numbers along the bottom refer to the SEQ IDnumber (see Table 13).

FIG. 32 depicts SDS/PAGE gel analysis of different C38S/C68 mutants thatremove a potential T cell epitope on C68S. R=Reduced sample.NR=Non-reduced sample

FIG. 33 depicts size-exclusion chromatography data from a shelfstability study for different C38S/C68 mutants that remove a potential Tcell epitope on C68S.

FIG. 34 depicts dose-response data using an HEK-Blue reporter cell line.All variants showed increased potency compared to WT IL-18 and similarpotency to the parent variant SEQ ID NO: 89.

FIG. 35 depicts size-exclusion chromatography data comparing SEQ ID NO:19 to SEQ ID NO: 89 from a Freeze-thaw study.

FIG. 36 depicts size-exclusion chromatography data comparing SEQ ID NO:19 to SEQ ID NO: 89 from an agitation study.

FIG. 37A and FIG. 37B depict results from surface plasmon resonanceexperiments to test affinity of SEQ ID NO: 19 and wild type human IL-18for human and cynomolgus monkey IL-18Rα (FIG. 37A) and IL-18BP (FIG.37B).

FIG. 38 depicts results from an IL-18BP resistance assay. WT hIL-18 waspotently inhibited by addition of IL-18BP, whereas SEQ ID NO: 19retained strong signaling capacity at all concentrations of IL-18BP.

FIG. 39 depicts tumor growth data in mice from dosing experiments (usingthe mouse DR IL-18 variant SEQ ID NO: 61).

FIG. 40 depicts tumor growth data in mice from additional dosingexperiments (using the mouse DR IL-18 variant SEQ ID NO: 61).

FIG. 41A and FIG. 41B depict dosing toleration experiments in primates(Cynomolgus macques) using a DR IL-18 variant (SEQ ID NO: 89 in thiscase).

FIG. 42A and FIG. 42B depicts production and purification of IL-18polypeptides (in this case DR IL-18 SEQ ID NO: 89) using cell-free SUMOprotease to cleave an N-terminal SUMO tag off of the IL-18 protein.

FIG. 43A and FIG. 43B depicts results from monitoring SUMO proteasecleavage by RP-HPLC.

FIG. 44 depicts production and purification of IL-18 polypeptides (inthis case DR IL-18 SEQ ID NO: 19) using exogenously provided SUMOprotease to cleave the N-terminal SUMO tag off of the IL-18 proteininside of a bacterial cell (in this case E. coli).

FIG. 45 depicts RP-HPLC data from monitoring the SUMO protease cleavagereaction inside of bacterial cells.

FIG. 46 depicts a Schematic of a yeast (e.g., Pichia pastoris) secretionsystem to produce IL-18 variants.

FIG. 47 depicts a representative reduced SDS-PAGE gel showing expressionof DR-18 (IL-18 variants—in this case SEQ ID NO: 89 and SEQ ID NO: 87.

FIG. 48 depicts a 2-chromatographic-step process (and associated data)that facilitated tagless purification of IL-18 from a yeast secretionexpression system (in this case P. Pastoris).

DETAILED DESCRIPTION OF THE INVENTION

Provided are variant (e.g., stabilized) IL-18 polypeptides and methodsof use (e.g., to treat a disease or disorder). A subject stabilizedIL-18 polypeptide includes mutations of two cysteine residues (C38 andC68) relative to the human wild type IL-18 (SEQ ID NO: 30). In somecases, the mutation is a C to S substitution and as such the stabilizedIL-18 polypeptide can in some cases comprise the mutations C38S andC68S. In some cases a subject stabilized IL-18 polypeptide comprises themutation pair C38S/C68S, C38S/C68G, C38S/C68A, C38S/C68V, C38S/C68D,C38S/C68E, or C38S/C68N (e.g., in some cases C38S/C68G, C38S/C68A,C38S/C68V, C38S/C68D, C38S/C68E, or C38S/C68N; in some cases C38S/C68S,C38S/C68G, C38S/C68A, C38S/C68D, or C38S/C68N; and in some casesC38S/C68G, C38S/C68A, C38S/C68D, or C38S/C68N).

In some cases, a subject stabilized IL-18 polypeptide is a stabilizedIL-18 variant polypeptide (e.g., a stabilized “decoy resistant” (DR)IL-18 variant or a stabilized “decoy-to-the-decoy” (D2D) IL-18 variant),i.e., an IL-18 variant that additionally includes mutations at positionsC38 and C68 relative to human wild type IL-18.

Provided are methods of administering an IL-18 polypeptide (e.g., a wildtype IL-18 or a variant such as a stabilized, DR, D2D, stabilized DR, orstabilized D2D IL-18 variant) to an individual (e.g., subcutaneously)where the frequency of administration is not more than once per week.Also provided are methods of producing/making a polypeptide. Some suchmethods include contacting an exogenously provided fusion protein insideof a cell (e.g., a bacterial cell) with a SUMO protease—where the fusionprotein includes a protein of interest fused at the N-terminus to a SUMOtag, whereby the SUMO protease cleaves the fusion protein to remove theSUMO tag from the protein of interest.

Before the present invention is described in greater detail, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges and are also encompassed within the invention, subject toany specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Certain ranges are presented herein with numerical values being precededby the term “about.” The term “about” is used herein to provide literalsupport for the exact number that it precedes, as well as a number thatis near to or approximately the number that the term precedes. Indetermining whether a number is near to or approximately a specificallyrecited number, the near or approximating unrecited number may be anumber which, in the context in which it is presented, provides thesubstantial equivalent of the specifically recited number.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, representativeillustrative methods and materials are now described.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention. Further, the dates ofpublication provided may be different from the actual publication dateswhich may need to be independently confirmed.

It is noted that, as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentinvention. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible.

While the apparatus and method has or will be described for the sake ofgrammatical fluidity with functional explanations, it is to be expresslyunderstood that the claims, unless expressly formulated under 35 U.S.C.§ 112, are not to be construed as necessarily limited in any way by theconstruction of “means” or “steps” limitations, but are to be accordedthe full scope of the meaning and equivalents of the definition providedby the claims under the judicial doctrine of equivalents, and in thecase where the claims are expressly formulated under 35 U.S.C. § 112 areto be accorded full statutory equivalents under 35 U.S.C. § 112

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. In describing and claiming thepresent invention, the following terminology will be used.

It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

“About” as used herein when referring to a measurable value such as anamount, a temporal duration, and the like, is meant to encompassnon-limiting variations of ±40% or ±20% or ±10%, ±5%, ±1%, or ±0.1% fromthe specified value, as such variations are appropriate.

The term “abnormal” when used in the context of organisms, tissues,cells or components thereof, refers to those organisms, tissues, cellsor components thereof that differ in at least one observable ordetectable characteristic (e.g., age, treatment, time of day, etc.) fromthose organisms, tissues, cells or components thereof that display the“normal” (expected) respective characteristic. Characteristics which arenormal or expected for one cell or tissue type, might be abnormal for adifferent cell or tissue type.

The term “antibody,” as used herein, refers to an immunoglobulinmolecule which is able to specifically bind to a specific epitope on anantigen. Antibodies can be intact immunoglobulins derived from naturalsources or from recombinant sources and can be immunoreactive portionsof intact immunoglobulins. The antibodies in the present invention mayexist in a variety of forms including, for example, polyclonalantibodies, monoclonal antibodies, intracellular antibodies(“intrabodies”), Fv, Fab and F(ab)2, as well as single chain antibodies(scFv), heavy chain antibodies, such as camelid antibodies, syntheticantibodies, chimeric antibodies, and a humanized antibodies (Harlow etal., 1999, Using Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory Press, NY; Harlow et al., 1989, Antibodies: A LaboratoryManual, Cold Spring Harbor, NY; Houston et al., 1988, Proc. Natl. Acad.Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).

An “antibody heavy chain,” as used herein, refers to the larger of thetwo types of polypeptide chains present in all antibody molecules intheir naturally occurring conformations.

An “antibody light chain,” as used herein, refers to the smaller of thetwo types of polypeptide chains present in all antibody molecules intheir naturally occurring conformations. κ and λ light chains refer tothe two major antibody light chain isotypes.

By the term “synthetic antibody” as used herein, is meant an antibodywhich is generated using recombinant DNA technology, such as, forexample, an antibody expressed by a bacteriophage as described herein.The term should also be construed to mean an antibody which has beengenerated by the synthesis of a DNA molecule encoding the antibody andwhich DNA molecule expresses an antibody protein, or an amino acidsequence specifying the antibody, wherein the DNA or amino acid sequencehas been obtained using synthetic DNA or amino acid sequence technologywhich is available and well known in the art.

As used herein, an “immunoassay” refers to any binding assay that usesan antibody capable of binding specifically to a target molecule todetect and quantify the target molecule.

By the term “specifically binds,” as used herein, e.g., with respect toan IL-18 variant polypeptide, is meant an IL-18 variant polypeptide thatrecognizes and binds to a specific molecule, such as IL-18R, or toIL-18BP. As an example, wild type IL-18 can be said to specifically bindto both IL-18R and to IL-18BP. In some instances, the IL-18 variantpolypeptide substantially reduced binding to IL-18BP. For example, anIL-18 variant polypeptide that specifically binds to a receptor from onespecies may also bind to that receptor from one or more species. But,such cross-species reactivity does not itself alter the classificationof an IL-18 variant polypeptide as specific. In another example, anIL-18 variant polypeptide that specifically binds to a receptor may alsobind to different allelic forms of the receptor. However, such crossreactivity does not itself alter the classification of an IL-18 variantpolypeptide as specific. In some instances, the terms “specific binding”or “specifically binding,” can be used in reference to the interactionof an antibody, a protein, or a peptide with a chemical species, to meanthat the interaction is dependent upon the presence of a particularstructure (e.g., an antigenic determinant or epitope) on the chemicalspecies; for example, an IL-18 variant polypeptide recognizes and bindsto a specific protein structure rather than to proteins generally.Similarly, the term “targets”, e.g., in the context of antibodies that“target” a particular antigen, is used to refer to the specific bindingpartner of a given molecule. For example, an agent (e.g., an antibody)that “targets” a particular protein/antigen specifically binds thatprotein/antigen in the sense that it preferentially binds to thatparticular protein/antigen over other proteins/antigens.

By the term “applicator,” as the term is used herein, is meant anydevice including, but not limited to, a hypodermic syringe, a pipette,an iontophoresis device, a patch, and the like, for administering thecompositions of the invention to a subject.

The term “coding sequence,” as used herein, means a sequence of anucleic acid or its complement, or a part thereof, that can betranscribed and/or translated to produce the mRNA and/or the polypeptideor a fragment thereof. Coding sequences include exons in a genomic DNAor immature primary RNA transcripts, which are joined together by thecell's biochemical machinery to provide a mature mRNA. The anti-sensestrand is the complement of such a nucleic acid, and the coding sequencecan be deduced therefrom. In contrast, the term “non-coding sequence,”as used herein, means a sequence of a nucleic acid or its complement, ora part thereof, that is not translated into amino acid in vivo, or wheretRNA does not interact to place or attempt to place an amino acid.Non-coding sequences include both intron sequences in genomic DNA orimmature primary RNA transcripts, and gene-associated sequences such aspromoters, enhancers, silencers, and the like.

As used herein, the terms “complementary” or “complementarity” are usedin reference to polynucleotides (i.e., a sequence of nucleotides)related by the base-pairing rules. For example, the sequence “A-G-T,” iscomplementary to the sequence “T-C-A.” Complementarity may be “partial,”in which only some of the nucleic acids' bases are matched according tothe base pairing rules. Or, there may be “complete” or “total”complementarity between the nucleic acids. The degree of complementaritybetween nucleic acid strands has significant effects on the efficiencyand strength of hybridization between nucleic acid strands. This is ofparticular importance in amplification reactions, as well as detectionmethods that depend upon binding between nucleic acids.

A “disease” is a state of health of an animal wherein the animal cannotmaintain homeostasis, and wherein if the disease is not ameliorated thenthe animal's health continues to deteriorate. In contrast, a “disorder”in an animal is a state of health in which the animal is able tomaintain homeostasis, but in which the animal's state of health is lessfavorable than it would been the absence of the disorder. Leftuntreated, a disorder does not necessarily cause a further decrease inthe animal's state of health.

An “effective amount” as used herein, means an amount which provides atherapeutic, prophylactic, or other desired benefit.

“Encoding” refers to the inherent property of specific sequences ofnucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, toserve as templates for synthesis of other polymers and macromolecules inbiological processes having either a defined sequence of nucleotides(i.e., guide RNA, rRNA, tRNA and mRNA) or a defined sequence of aminoacids and the biological properties resulting therefrom. Thus, a geneencodes a protein if transcription and translation of mRNA correspondingto that gene produces the protein in a cell or other biological system.Both the coding strand, the nucleotide sequence of which is identical tothe mRNA sequence and is usually provided in sequence listings, and thenon-coding strand, used as the template for transcription of a gene orcDNA, can be referred to as encoding the protein or other product ofthat gene or cDNA.

As used herein, the term “fragment,” as applied to a nucleic acid,refers to a subsequence of a larger nucleic acid. A “fragment” of anucleic acid can be at least about 15 nucleotides in length; forexample, at least about 50 nucleotides to about 100 nucleotides; atleast about 100 to about 500 nucleotides, at least about 500 to about1000 nucleotides; at least about 1000 nucleotides to about 1500nucleotides; about 1500 nucleotides to about 2500 nucleotides; or about2500 nucleotides (and any integer value in between). As used herein, theterm “fragment,” as applied to a protein, polypeptide or peptide, refersto a subsequence of a larger protein, polypeptide or peptide. A“fragment” of a protein, polypeptide, or peptide can be at least about 5amino acids in length; for example, at least about 10 amino acids inlength; at least about 20 amino acids in length; at least about 50 aminoacids in length; at least about 100 amino acids in length; at leastabout 200 amino acids in length; or at least about 300 amino acids inlength (and any integer value in between).

The term “gene” refers to a nucleic acid (e.g., DNA) sequence thatincludes coding sequences necessary for the production of a polypeptide,precursor, or RNA (e.g., mRNA). The polypeptide may be encoded by afull-length coding sequence or by any portion of the coding sequence solong as the desired activity or functional property (e.g., enzymaticactivity, receptor binding, signal transduction, immunogenicity, etc.)of the full-length or fragment is retained. The term also encompassesthe coding region of a structural gene and the sequences locatedadjacent to the coding region on both the 5′ and 3′ ends for a distanceof about 2 kb or more on either end such that the gene corresponds tothe length of the full-length mRNA and 5′ regulatory sequences whichinfluence the transcriptional properties of the gene. Sequences located5′ of the coding region and present on the mRNA are referred to as5′-untranslated sequences. The 5′-untranslated sequences usually containthe regulatory sequences. Sequences located 3′ or downstream of thecoding region and present on the mRNA are referred to as 3′-untranslatedsequences. The term “gene” encompasses both cDNA and genomic forms of agene. A genomic form or clone of a gene contains the coding regioninterrupted with non-coding sequences termed “introns” or “interveningregions” or “intervening sequences.” Introns are segments of a gene thatare transcribed into nuclear RNA (hnRNA); introns may contain regulatoryelements such as enhancers. Introns are removed or “spliced out” fromthe nuclear or primary transcript; introns therefore are absent in themessenger RNA (mRNA) transcript. The mRNA functions during translationto specify the sequence or order of amino acids in a nascentpolypeptide.

“Homologous”, “identical,” or “identity” as used herein in the contextof two or more nucleic acids or polypeptide sequences means that thesequences have a specified percentage of residues that are the same overa specified region. The percentage can be calculated by optimallyaligning the two sequences, comparing the two sequences over thespecified region, determining the number of positions at which theidentical residue occurs in both sequences to yield the number ofmatched positions, dividing the number of matched positions by the totalnumber of positions in the specified region, and multiplying the resultby 100 to yield the percentage of sequence identity. In cases where thetwo sequences are of different lengths or the alignment produces one ormore staggered ends and the specified region of comparison includes onlya single sequence, the residues of the single sequence are included inthe denominator but not the numerator of the calculation. When comparingDNA and RNA, thymine (T) and uracil (U) can be considered equivalent.Identity can be performed manually or by using a computer sequencealgorithm such as BLAST or BLAST 2.0.

“Instructional material,” as that term is used herein, includes apublication, a recording, a diagram, or any other medium of expressionwhich can be used to communicate the usefulness of the nucleic acid,peptide, polypeptide, and/or compound of the invention in the kit foridentifying or alleviating or treating the various diseases or disordersrecited herein. Optionally, or alternately, the instructional materialmay describe one or more methods of identifying or alleviating thediseases or disorders in a cell or a tissue of a subject. Theinstructional material of the kit may, for example, be affixed to acontainer that contains the nucleic acid, polypeptide, and/or compoundof the invention or be shipped together with a container that containsthe nucleic acid, polypeptide, and/or compound. Alternatively, theinstructional material may be shipped separately from the container withthe intention that the recipient uses the instructional material and thecompound cooperatively.

“Isolated” means altered or removed from the natural state. For example,a nucleic acid or a polypeptide naturally present in a living animal isnot “isolated,” but the same nucleic acid or polypeptide partially orcompletely separated from the coexisting materials of its natural stateis isolated.” An isolated nucleic acid or protein can exist insubstantially purified form, or can exist in a non-native environmentsuch as, for example, a host cell.

As used herein, the terms “purify” and “purified” in the context of aprotein refers to level of purity that allows for the effective use ofthe protein, e.g., in vitro, ex vivo, or in vivo. For a protein to beuseful for a given application, it should be substantially free ofcontaminants, other proteins, and/or chemicals that could interfere withthe use of that protein in such application, or that at least would beundesirable for inclusion with the protein of interest. Suchapplications include that preparation of therapeutic compositions, theadministration of the protein in a therapeutic composition, and othermethods disclosed herein. Preferably, a “purified” protein, asreferenced herein, is a protein that can be produced by any method(i.e., by direct purification from a natural source, recombinantly, orsynthetically), and that has been purified from other protein componentssuch that the protein comprises at least about 75% weight/weight of thetotal protein in a given composition, 80% weight/weight of the totalprotein in a given composition, and more preferably, at least about 85%,and more preferably at least about 90%, and more preferably at leastabout 91%, and more preferably at least about 92%, and more preferablyat least about 93%, and more preferably at least about 94%, and morepreferably at least about 95%, and more preferably at least about 96%,and more preferably at least about 97%, and more preferably at leastabout 98%, and more preferably at least about 99% weight/weight of thetotal protein in a given composition. As an example, a purifiedpolypeptide is a polypeptide which has been separated from othercomponents with which it might normally be associated in its naturallyoccurring state (e.g., if the protein is a naturally existing protein)and from components with which it may be associated while inside of acell or in extracellular milieu. For example, in some cases a proteincan be purified from a cellular lysate (e.g., from a lysate of bacterialcells in which the protein was exogenously expressed). As anotherexample a protein can, be purified from an extracellular medium, e.g.,from culture medium into which cells (e.g., yeast cells) have secretedthe protein.

An “isolated nucleic acid” refers to a nucleic acid segment or fragmentwhich has been separated from sequences which flank it in a naturallyoccurring state, e.g., a DNA fragment which has been removed from thesequences which are normally adjacent to the fragment, e.g., thesequences adjacent to the fragment in a genome in which it naturallyoccurs. The term also applies to nucleic acids which have beensubstantially purified from other components which naturally accompanythe nucleic acid, e.g., RNA or DNA or proteins, which naturallyaccompany it in the cell. The term therefore includes, for example, arecombinant DNA which is incorporated into a vector, into anautonomously replicating plasmid or virus, or into the genomic DNA of aprokaryote or eukaryote, or which exists as a separate molecule (e.g.,as a cDNA or a genomic or cDNA fragment produced by PCR or restrictionenzyme digestion) independent of other sequences. It also includes arecombinant DNA which is part of a hybrid gene encoding additionalpolypeptide sequence.

The term “label” when used herein refers to a detectable compound orcomposition that is conjugated directly or indirectly to a probe togenerate a “labeled” probe. The label may be detectable by itself (e.g.,radioisotope labels or fluorescent labels) or, in the case of anenzymatic label, may catalyze chemical alteration of a substratecompound or composition that is detectable (e.g., avidin-biotin). Insome instances, primers can be labeled to detect a PCR product.

By the term “modulating,” as used herein, is meant mediating adetectable increase or decrease in the activity and/or level of a mRNA,polypeptide, or a response in a subject compared with the activityand/or level of the mRNA, polypeptide or response in the subject in theabsence of a treatment or compound, and/or compared with the activityand/or level of the mRNA, polypeptide, or response in an otherwiseidentical but untreated subject. The term encompasses activating,inhibiting and/or otherwise affecting a native signal or responsethereby mediating a beneficial therapeutic, prophylactic, or otherdesired response in a subject, for example, a human. A “mutation,”“mutant,” or “variant,” as used herein, refers to a change in nucleicacid or amino acid sequence relative to a reference sequence (which maybe a naturally-occurring normal/“wild-type” sequence), and includestranslocations, deletions, insertions, and substitutions/pointmutations. A “mutant” or “variant” as used herein, refers to either anucleic acid or protein comprising a mutation.

A “nucleic acid” refers to a polynucleotide and includespoly-ribonucleotides and poly-deoxyribonucleotides. Nucleic acidsaccording to the present invention may include any polymer or oligomerof pyrimidine and purine bases, preferably cytosine, thymine, anduracil, and adenine and guanine, respectively. (See Albert L. Lehninger,Principles of Biochemistry, at 793-800 (Worth Pub. 1982) which is hereinincorporated in its entirety for all purposes). Indeed, the presentinvention contemplates any deoxyribonucleotide, ribonucleotide orpeptide nucleic acid component, and any chemical variants thereof, suchas methylated, hydroxymethylated or glucosylated forms of these bases,and the like. The polymers or oligomers may be heterogeneous orhomogeneous in composition, and may be isolated from naturally occurringsources or may be artificially or synthetically produced. In addition,the nucleic acids may be DNA or RNA, or a mixture thereof, and may existpermanently or transitionally in single-stranded or double-strandedform, including homoduplex, heteroduplex, and hybrid states.

An “oligonucleotide” or “polynucleotide” is a nucleic acid ranging fromat least 2, preferably at least 8, 15 or 25 nucleotides in length, butmay be up to 50, 100, 1000, or 5000 nucleotides long or a compound thatspecifically hybridizes to a polynucleotide. Polynucleotides includesequences of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) ormimetics thereof which may be isolated from natural sources,recombinantly produced or artificially synthesized. A further example ofa polynucleotide of the present invention may be a peptide nucleic acid(PNA). (See U.S. Pat. No. 6,156,501 which is hereby incorporated byreference in its entirety). The invention also encompasses situations inwhich there is a nontraditional base pairing such as Hoogsteen basepairing which has been identified in certain tRNA molecules andpostulated to exist in a triple helix. “Polynucleotide” and“oligonucleotide” are used interchangeably in this disclosure. It willbe understood that when a nucleotide sequence is represented herein by aDNA sequence (e.g., A, T, G, and C), this also includes thecorresponding RNA sequence (e.g., A, U, G, C) in which “U” replaces “T”.

The terms “individual,” “subject,” “host,” and “patient,” are usedinterchangeably herein and refer to any mammalian subject for whomdiagnosis, treatment, or therapy is desired, particularly humans.

As used herein, the terms “peptide,” “polypeptide,” and “protein” areused interchangeably, and refer to a compound comprised of amino acidresidues covalently linked by peptide bonds. A protein or peptide mustcontain at least two amino acids, and no limitation is placed on themaximum number of amino acids that can comprise a protein's or peptide'ssequence. Polypeptides include any peptide or protein comprising two ormore amino acids joined to each other by peptide bonds. As used herein,the term refers to both short chains, which also commonly are referredto in the art as peptides, oligopeptides and oligomers, for example, andto longer chains, which generally are referred to in the art asproteins, of which there are many types. “Polypeptides” include, forexample, biologically active fragments, substantially homologouspolypeptides, oligopeptides, homodimers, heterodimers, variants ofpolypeptides, modified polypeptides, derivatives, analogs, fusionproteins, among others. The polypeptides include natural peptides,recombinant peptides, synthetic peptides, mutant polypeptides, variantpolypeptides, or a combination thereof.

As used herein, “polynucleotide” includes cDNA, RNA, DNA/RNA hybrid,antisense RNA, ribozyme, genomic DNA, synthetic forms, and mixedpolymers, both sense and antisense strands, and may be chemically orbiochemically modified to exhibit non-natural or derivatized, synthetic,or semi-synthetic nucleotide bases. Also, contemplated are alterationsof a wild type or synthetic gene, including but not limited to deletion,insertion, substitution of one or more nucleotides, or fusion to otherpolynucleotide sequences.

“Sample” or “biological sample” as used herein means a biologicalmaterial isolated from a subject. The biological sample may contain anybiological material suitable for detecting a mRNA, polypeptide or othermarker of a physiologic or pathologic process in a subject, and maycomprise fluid, tissue, cellular and/or non-cellular material obtainedfrom the individual.

As used herein, the terms “therapy” or “therapeutic regimen” refer tothose activities taken to prevent, treat or alter a disease or disorder,e.g., a course of treatment intended to reduce or eliminate at least onesign or symptom of a disease or disorder using pharmacological,surgical, dietary and/or other techniques. A therapeutic regimen mayinclude a prescribed dosage of one or more compounds or surgery.Therapies will most often be beneficial and reduce or eliminate at leastone sign or symptom of the disorder or disease state, but in someinstances the effect of a therapy will have non-desirable orside-effects. The effect of therapy will also be impacted by thephysiological state of the subject, e.g., age, gender, genetics, weight,other disease conditions, etc.

The term “therapeutically effective amount” refers to the amount of thesubject compound or composition that will elicit the intendedbiological, physiologic, clinical or medical response of a cell, tissue,organ, system, or subject that is being sought by the researcher,veterinarian, medical doctor or other clinician. The term“therapeutically effective amount” includes that amount of a compound orcomposition that, when administered, is sufficient to treat one or moreof the signs or symptoms of the disorder or disease being treated. Thetherapeutically effective amount will vary depending on the compound orcomposition, the disease and its severity and the age, weight, etc., ofthe subject to be treated.

To “treat” a disease or disorder as the term is used herein, means toreduce the frequency or severity of at least one sign or symptom of adisease or disorder experienced by a subject. The terms “treatment”,“treating”, “treat” and the like are used herein to generally refer toobtaining a desired pharmacologic and/or physiologic effect. The effectcan be prophylactic in terms of completely or partially preventing adisease or symptom(s) thereof and/or may be therapeutic in terms of apartial or complete stabilization or cure for a disease and/or adverseeffect attributable to the disease. The term “treatment” encompasses anytreatment of a disease in a mammal, particularly a human, and includes:(a) preventing the disease and/or symptom(s) from occurring in a subjectwho may be predisposed to the disease or symptom but has not yet beendiagnosed as having it; (b) inhibiting the disease and/or symptom(s),e.g., slowing or arresting their development (e.g., halting the growthof tumors, slowing the rate of tumor growth, halting the rate of cancercell proliferation, and the like); or (c) relieving the diseasesymptom(s), i.e., causing regression of the disease and/or symptom(s)(e.g., causing decrease in tumor size, reducing the number of cancercells present, and the like). Those in need of treatment include thosealready inflicted (e.g., those with cancer, those with an infection,those with a metabolic disorder, those with macular degeneration, etc.)as well as those in which prevention is desired (e.g., those withincreased susceptibility to cancer, those with an increased likelihoodof infection, those suspected of having cancer, those suspected ofharboring an infection, those with increased susceptibility formetabolic disease, those with increased susceptibility for maculardegeneration, etc.).

As used herein, the term “wild-type” refers to a gene or gene productisolated from a naturally occurring source or having a naturallyoccurring sequence (e.g., a wild type protein with a naturally occurringamino acid sequence can be isolated from a natural source or from asynthetic source, but would still be considered a wild type protein). Incontrast, the term “modified,” “variant,” or “mutant” refers to a geneor gene product that possesses modifications in sequence and/orfunctional properties (i.e., altered characteristics) when compared tothe wild type gene or gene product.

Ranges: throughout this disclosure, various aspects can be presented ina range format. It should be understood that the description in rangeformat is merely for convenience and brevity and should not be construedas an inflexible limitation on the scope of the invention. Accordingly,the description of a range should be considered to have specificallydisclosed all the possible subranges as well as individual numericalvalues within that range. For example, description of a range such asfrom 1 to 6 should be considered to have specifically disclosedsubranges such as from 1 to 2, 1 to 3, 1 to 4, 1 to 5, 2 to 3, 2 to 4, 2to 5, 2 to 6, etc., as well as individual numbers within that range, forexample, 1, 2, 2.7. 3, 4, 5, 5.3, and 6. This applies regardless of thebreadth of the range.

Compositions and Methods

As summarized above, in some embodiments, the compositions and methodsof the disclosure comprise an IL-18 polypeptide having mutations atamino acid positions C38 and C68 relative to wild type IL-18 (SEQ ID NO:30). Such variants are referred to herein as “stabilized” IL-18variants.

In some embodiments, the compositions and methods of the disclosurecomprise an IL-18 variant polypeptide that is mutated relative to wildtype IL-18 such that it can bind to one of IL-18's natural bindingpartners, but not to the other (or has reduced binding to the other).For example, wild type IL-18 binds to both IL-18R (to signal through thereceptor) and IL-18BP (which inhibits IL-18 by preventing it frombinding to IL-18R). In some case an IL-18 variant polypeptide discussedherein is a “decoy resistant IL-18” (“DR IL-18”) variant; such variantsbind to IL-18R but have reduced binding to (and in some cases do notbind to) IL-18BP. In other cases an IL-18 variant polypeptide discussedherein is a “decoy-to-the-decoy IL-18” (“D2D IL-18”) variant; suchvariants bind to IL-18BP but have reduced binding to (and in some casesdo not bind to) IL-18R. As shown in this disclosure, an IL-18 variantpolypeptide may show comparable binding affinity to IL-18Rα as wild typeIL-18, while also exhibiting decreased binding affinity to IL-18BPcompared to wild type IL-18.

In some cases, there is overlap between the terms to describe thevarious variants. For example, a DR IL-18 variant or D2D IL-18 variantcan include mutations at C38 and C68 and in such cases the IL-18variants would be “stabilized” variants. As such, the term “stabilized”encompasses stabilized wild type IL-18 (WT IL-18 with C38 and C68mutations) as well as stabilized variant proteins such as DR IL-18 andD2D IL-18 variants that have C38 and C68 mutations. Likewise, the term“DR IL-18” variant encompasses such variants with and without C38/C68mutations and the term “D2D IL-18” variant encompasses such variantswith and without C38/C68 mutations. As such, the term “stabilized DRIL-18” variant can be used when discussing DR IL-18 variants that haveC38/C68 mutations and the term stabilized D2D IL-18″ variant can be usedwhen discussing D2D IL-18 variants that have C38/C68 mutations.

As such, the following will discuss various embodiments of “stabilized”IL-18 proteins (various C38/C68 mutants). This description equallyapplies to any type of IL-18 protein (e.g., wild type, DR IL-18 variant,D2D IL-18 variant, etc.). Mutations in C38/C68 are first discussed,followed by a discussion of DR IL-18 variants and D2D IL-18 variants.All of the discussed variants can be used in any of the methodsdescribed herein.

A stabilized IL-18 polypeptide is an IL-18 polypeptide that includes‘stabilizing mutations’ which are mutations of two cysteine residues(C38 and C68) relative to the human wild type IL-18 (SEQ ID NO: 30).Surprisingly, mutation of these two amino acids was found to stabilizeIL-18 better than all other possible combinations of Cysteine mutations,see, e.g., the working examples below. In some cases, the mutation is aC to S substitution and as such the stabilized IL-18 polypeptide can insome cases comprise the mutations C38S and C68S (also referred to hereinas “mutation pair C38S/C68S”) relative to the human wild type IL-18 (SEQID NO: 30). In some cases, a subject IL-18 polypeptide includes an aminoacid sequence having 80% or more sequence identity (e.g., 85% or more,90% or more, 92% or more, 95% or more, 97% or more, 98% or more, or98.5% or more sequence identity) with human wild type IL-18 (SEQ ID NO:30) and includes mutations at amino acid positions C38 and C68 relativeto SEQ ID NO: 30. In some cases, a subject IL-18 polypeptide includes anamino acid sequence having 90% or more sequence identity (e.g., 92% ormore, 95% or more, 97% or more, 98% or more, or 98.5% or more sequenceidentity) with human wild type IL-18 (SEQ ID NO: 30) and includesmutations at amino acid positions C38 and C68 relative to SEQ ID NO: 30.Examples of such variant proteins include those set forth as SEQ ID NOs:6 and 16-21, although these sequences also include additional mutationsthat render them DR IL-18 variants (See below)—and such SEQ ID NOs: 6and 16-21 are examples of stabilized DR IL-18 variants.

In some cases the mutation is a C to S substitution and as such thestabilized IL-18 polypeptide can in some cases comprise the mutationsC38S and C68S (also referred to herein as “C38S/C68S”) relative to thehuman wild type IL-18 (SEQ ID NO: 30). Mutation of C68 can in some casesresult in an immunogenic epitope. Thus, in some embodiments the mutationat C68 is a non-immunogenic substitution. In some such cases themutation is a C68 to G, A, V, D, E, or N mutation (in some cases C68 toG, A, D, or N). Thus, in some such cases a subject stabilized IL-18polypeptide comprises the mutations C38S/C68S, C38S/C68G, C38S/C68A,C38S/C68V, C38S/C68D, C38S/C68E, or C38S/C68N. In some cases, a subjectstabilized IL-18 polypeptide comprises the mutations C38S/C68G,C38S/C68A, C38S/C68V, C38S/C68D, C38S/C68E, or C38S/C68N. In some cases,a subject stabilized IL-18 polypeptide comprises the mutationsC38S/C68S, C38S/C68G, C38S/C68A, C38S/C68D, or C38S/C68N. In some cases,a subject stabilized IL-18 polypeptide comprises the mutationsC38S/C68G, C38S/C68A, C38S/C68D, or C38S/C68N. In some cases, a subjectstabilized IL-18 polypeptide comprises the mutations C38S/C68D. In somecases, a subject stabilized IL-18 polypeptide comprises the mutationsC38S/C68G. In some cases, a subject stabilized IL-18 polypeptidecomprises the mutations C38S/C68A. In some cases, a subject stabilizedIL-18 polypeptide comprises the mutations C38S/C68A. In some cases, asubject stabilized IL-18 polypeptide comprises the mutations C38S/C68N.

Thus, in some embodiments, a subject IL-18 polypeptide includes an aminoacid sequence having 80% or more sequence identity (e.g., 85% or more,90% or more, 92% or more, 95% or more, 97% or more, 98% or more, or98.5% or more sequence identity) with human wild type IL-18 (SEQ ID NO:30) and includes the mutations C38S/C68S, C38S/C68G, C38S/C68A,C38S/C68V, C38S/C68D, C38S/C68E, or C38S/C68N relative to SEQ ID NO: 30.In some embodiments, a subject IL-18 polypeptide includes an amino acidsequence having 80% or more sequence identity (e.g., 85% or more, 90% ormore, 92% or more, 95% or more, 97% or more, 98% or more, or 98.5% ormore sequence identity) with human wild type IL-18 (SEQ ID NO: 30) andincludes the mutations C38S/C68G, C38S/C68A, C38S/C68V, C38S/C68D,C38S/C68E, or C38S/C68N relative to SEQ ID NO: 30. In some embodiments,a subject IL-18 polypeptide includes an amino acid sequence having 80%or more sequence identity (e.g., 85% or more, 90% or more, 92% or more,95% or more, 97% or more, 98% or more, or 98.5% or more sequenceidentity) with human wild type IL-18 (SEQ ID NO: 30) and includes themutations C38S/C68S, C38S/C68G, C38S/C68A, C38S/C68D, or C38S/C68Nrelative to SEQ ID NO: 30. In some embodiments, a subject IL-18polypeptide includes an amino acid sequence having 80% or more sequenceidentity (e.g., 85% or more, 90% or more, 92% or more, 95% or more, 97%or more, 98% or more, or 98.5% or more sequence identity) with humanwild type IL-18 (SEQ ID NO: 30) and includes the mutations C38S/C68G,C38S/C68A, C38S/C68D, or C38S/C68N relative to SEQ ID NO: 30. In someembodiments, a subject IL-18 polypeptide includes an amino acid sequencehaving 80% or more sequence identity (e.g., 85% or more, 90% or more,92% or more, 95% or more, 97% or more, 98% or more, or 98.5% or moresequence identity) with human wild type IL-18 (SEQ ID NO: 30) andincludes the mutations C38S/C68D relative to SEQ ID NO: 30. In someembodiments, a subject IL-18 polypeptide includes an amino acid sequencehaving 80% or more sequence identity (e.g., 85% or more, 90% or more,92% or more, 95% or more, 97% or more, 98% or more, or 98.5% or moresequence identity) with human wild type IL-18 (SEQ ID NO: 30) andincludes the mutations C38S/C68G relative to SEQ ID NO: 30. In someembodiments, a subject IL-18 polypeptide includes an amino acid sequencehaving 80% or more sequence identity (e.g., 85% or more, 90% or more,92% or more, 95% or more, 97% or more, 98% or more, or 98.5% or moresequence identity) with human wild type IL-18 (SEQ ID NO: 30) andincludes the mutations C38S/C68A relative to SEQ ID NO: 30. In someembodiments, a subject IL-18 polypeptide includes an amino acid sequencehaving 80% or more sequence identity (e.g., 85% or more, 90% or more,92% or more, 95% or more, 97% or more, 98% or more, or 98.5% or moresequence identity) with human wild type IL-18 (SEQ ID NO: 30) andincludes the mutations C38S/C68N relative to SEQ ID NO: 30.

In some embodiments, a subject IL-18 polypeptide includes an amino acidsequence having 90% or more sequence identity (e.g., 92% or more, 95% ormore, 97% or more, 98% or more, or 98.5% or more sequence identity) withhuman wild type IL-18 (SEQ ID NO: 30) and includes the mutationsC38S/C68S, C38S/C68G, C38S/C68A, C38S/C68V, C38S/C68D, C38S/C68E, orC38S/C68N relative to SEQ ID NO: 30. In some embodiments, a subjectIL-18 polypeptide includes an amino acid sequence having 90% or moresequence identity (e.g., 92% or more, 95% or more, 97% or more, 98% ormore, or 98.5% or more sequence identity) with human wild type IL-18(SEQ ID NO: 30) and includes the mutations C38S/C68G, C38S/C68A,C38S/C68V, C38S/C68D, C38S/C68E, or C38S/C68N relative to SEQ ID NO: 30.In some embodiments, a subject IL-18 polypeptide includes an amino acidsequence having 90% or more sequence identity (e.g., 92% or more, 95% ormore, 97% or more, 98% or more, or 98.5% or more sequence identity) withhuman wild type IL-18 (SEQ ID NO: 30) and includes the mutationsC38S/C68S, C38S/C68G, C38S/C68A, C38S/C68D, or C38S/C68N relative to SEQID NO: 30. In some embodiments, a subject IL-18 polypeptide includes anamino acid sequence having 90% or more sequence identity (e.g., 92% ormore, 95% or more, 97% or more, 98% or more, or 98.5% or more sequenceidentity) with human wild type IL-18 (SEQ ID NO: 30) and includes themutations C38S/C68G, C38S/C68A, C38S/C68D, or C38S/C68N relative to SEQID NO: 30. In some embodiments, a subject IL-18 polypeptide includes anamino acid sequence having 90% or more sequence identity (e.g., 92% ormore, 95% or more, 97% or more, 98% or more, or 98.5% or more sequenceidentity) with human wild type IL-18 (SEQ ID NO: 30) and includes themutations C38S/C68D relative to SEQ ID NO: 30. In some embodiments, asubject IL-18 polypeptide includes an amino acid sequence having 90% ormore sequence identity (e.g., 92% or more, 95% or more, 97% or more, 98%or more, or 98.5% or more sequence identity) with human wild type IL-18(SEQ ID NO: 30) and includes the mutations C38S/C68G relative to SEQ IDNO: 30. In some embodiments, a subject IL-18 polypeptide includes anamino acid sequence having 90% or more sequence identity (e.g., 92% ormore, 95% or more, 97% or more, 98% or more, or 98.5% or more sequenceidentity) with human wild type IL-18 (SEQ ID NO: 30) and includes themutations C38S/C68A relative to SEQ ID NO: 30. In some embodiments, asubject IL-18 polypeptide includes an amino acid sequence having 90% ormore sequence identity (e.g., 92% or more, 95% or more, 97% or more, 98%or more, or 98.5% or more sequence identity) with human wild type IL-18(SEQ ID NO: 30) and includes the mutations C38S/C68N relative to SEQ IDNO: 30.

Stabilized DR IL-18 or D2D IL-18 Variants

In some cases, a subject stabilized IL-18 polypeptide is a stabilizedIL-18 variant polypeptide, i.e., an IL-18 variant polypeptide thatincludes the stabilizing mutations (i.e., mutations at positions C38 andC68 relative to human wild type IL-18). In some such cases thestabilized IL-18 variant polypeptide is a stabilized “decoy resistant”(DR) IL-18 variant, and in other such cases the stabilized IL-18 variantpolypeptide is a stabilized “decoy-to-the-decoy” (D2D) IL-18 variant.Examples of human DR IL-18 variants include but are not limited to thoseset forth in SEQ ID NOs: 34-59, 73-91, and 191-193, while examples ofhuman D2D IL-18 variants include but are not limited to those set forthin SEQ ID NOs: 92-125; also see the working examples below. DR-IL18variants are described in detail below and any of such variant caninclude a C38/C68 mutation as described herein in order to be astabilized DR-IL18.

In some cases, a subject stabilized DR IL-18 variant comprises the aminoacid sequence set forth in SEQ ID NO: 89, except the C38 and C68 aremutated (e.g., C38S/C68S, C38S/C68G, C38S/C68A, C38S/C68V, C38S/C68D,C38S/C68E, or C38S/C68N—in some cases C38S/C68G, C38S/C68A, C38S/C68V,C38S/C68D, C38S/C68E, or C38S/C68N—in some cases C38S/C68S, C38S/C68G,C38S/C68A, C38S/C68D, or C38S/C68N—in some cases C38S/C68G, C38S/C68A,C38S/C68D, or C38S/C68N—in some cases C38S/C68D—in some casesC38S/C68G—in some cases C38S/C68A—in some cases C38S/C68N). Examples ofstabilized DR IL-18 variants include those set forth as SEQ ID NOs: 6and 16-21 (these are C38/C68 mutated versions of the DR-18 variant setforth as SEQ ID NO: 89). One of ordinary skill in the art would readilyrecognize that equivalent stabilized variants can easily be produced forany desired IL-18 variant (see, e.g., the working examples below) (e.g.,any of the human DR-IL variants set forth as SEQ ID NOs: 34-59, 73-91,and 191-193, e.g., SEQ ID NOs 87-91, or for any of the human D2D IL-18variants set forth as SEQ ID NOs: 92-125).

Thus, in some embodiments, a subject IL-18 polypeptide includes an aminoacid sequence having 80% or more sequence identity (e.g., 85% or more,90% or more, 92% or more, 95% or more, 97% or more, 98% or more, or98.5% or more sequence identity) with human wild type IL-18 (SEQ ID NO:30) [or any one of SEQ ID NOs: 6 and 16-21 or any one of SEQ ID NOs:34-59, 73-91, and 191-193 or any one of SEQ ID NOs:87-91 or SEQ ID NO:89] and includes the mutations C38S/C68S, C38S/C68G, C38S/C68A,C38S/C68V, C38S/C68D, C38S/C68E, or C38S/C68N relative to SEQ ID NO: 30.In some embodiments, a subject IL-18 polypeptide includes an amino acidsequence having 80% or more sequence identity (e.g., 85% or more, 90% ormore, 92% or more, 95% or more, 97% or more, 98% or more, or 98.5% ormore sequence identity) with human wild type IL-18 (SEQ ID NO: 30) [orany one of SEQ ID NOs: 6 and 16-21 or any one of SEQ ID NOs: 34-59,73-91, and 191-193 or any one of SEQ ID NOs:87-91 or SEQ ID NO: 89] andincludes the mutations C38S/C68G, C38S/C68A, C38S/C68V, C38S/C68D,C38S/C68E, or C38S/C68N relative to SEQ ID NO: 30. In some embodiments,a subject IL-18 polypeptide includes an amino acid sequence having 80%or more sequence identity (e.g., 85% or more, 90% or more, 92% or more,95% or more, 97% or more, 98% or more, or 98.5% or more sequenceidentity) with human wild type IL-18 (SEQ ID NO: 30) [or any one of SEQID NOs: 6 and 16-21 or any one of SEQ ID NOs: 34-59, 73-91, and 191-193or any one of SEQ ID NOs:87-91 or SEQ ID NO: 89] and includes themutations C38S/C68S, C38S/C68G, C38S/C68A, C38S/C68D, or C38S/C68Nrelative to SEQ ID NO: 30. In some embodiments, a subject IL-18polypeptide includes an amino acid sequence having 80% or more sequenceidentity (e.g., 85% or more, 90% or more, 92% or more, 95% or more, 97%or more, 98% or more, or 98.5% or more sequence identity) with humanwild type IL-18 (SEQ ID NO: 30) [or any one of SEQ ID NOs: 6 and 16-21or any one of SEQ ID NOs: 34-59, 73-91, and 191-193 or any one of SEQ IDNOs:87-91 or SEQ ID NO: 89] and includes the mutations C38S/C68G,C38S/C68A, C38S/C68D, or C38S/C68N relative to SEQ ID NO: 30. In someembodiments, a subject IL-18 polypeptide includes an amino acid sequencehaving 80% or more sequence identity (e.g., 85% or more, 90% or more,92% or more, 95% or more, 97% or more, 98% or more, or 98.5% or moresequence identity) with human wild type IL-18 (SEQ ID NO: 30) [or anyone of SEQ ID NOs: 6 and 16-21 or any one of SEQ ID NOs: 34-59, 73-91,and 191-193 or any one of SEQ ID NOs:87-91 or SEQ ID NO: 89] andincludes the mutations C38S/C68D relative to SEQ ID NO: 30.

In some embodiments, a subject IL-18 polypeptide includes an amino acidsequence having 80% or more sequence identity (e.g., 85% or more, 90% ormore, 92% or more, 95% or more, 97% or more, 98% or more, or 98.5% ormore sequence identity) with human wild type IL-18 (SEQ ID NO: 30) [orany one of SEQ ID NOs: 6 and 16-21 or any one of SEQ ID NOs: 34-59,73-91, and 191-193 or any one of SEQ ID NOs:87-91 or SEQ ID NO: 89] andincludes the mutations C38S/C68G relative to SEQ ID NO: 30. In someembodiments, a subject IL-18 polypeptide includes an amino acid sequencehaving 80% or more sequence identity (e.g., 85% or more, 90% or more,92% or more, 95% or more, 97% or more, 98% or more, or 98.5% or moresequence identity) with human wild type IL-18 (SEQ ID NO: 30) [or anyone of SEQ ID NOs: 6 and 16-21 or any one of SEQ ID NOs: 34-59, 73-91,and 191-193 or any one of SEQ ID NOs:87-91 or SEQ ID NO: 89] andincludes the mutations C38S/C68A relative to SEQ ID NO: 30.

In some embodiments, a subject IL-18 polypeptide includes an amino acidsequence having 80% or more sequence identity (e.g., 85% or more, 90% ormore, 92% or more, 95% or more, 97% or more, 98% or more, or 98.5% ormore sequence identity) with human wild type IL-18 (SEQ ID NO: 30) [orany one of SEQ ID NOs: 6 and 16-21 or any one of SEQ ID NOs: 34-59,73-91, and 191-193 or any one of SEQ ID NOs:87-91 or SEQ ID NO: 89] andincludes the mutations C38S/C68N relative to SEQ ID NO: 30. In someembodiments, a subject IL-18 polypeptide includes an amino acid sequencehaving 90% or more sequence identity (e.g., 92% or more, 95% or more,97% or more, 98% or more, or 98.5% or more sequence identity) with humanwild type IL-18 (SEQ ID NO: 30) [or any one of SEQ ID NOs: 6 and 16-21or any one of SEQ ID NOs: 34-59, 73-91, and 191-193 or any one of SEQ IDNOs:87-91 or SEQ ID NO: 89] and includes the mutations C38S/C68S,C38S/C68G, C38S/C68A, C38S/C68V, C38S/C68D, C38S/C68E, or C38S/C68Nrelative to SEQ ID NO: 30. In some embodiments, a subject IL-18polypeptide includes an amino acid sequence having 90% or more sequenceidentity (e.g., 92% or more, 95% or more, 97% or more, 98% or more, or98.5% or more sequence identity) with human wild type IL-18 (SEQ ID NO:30) [or any one of SEQ ID NOs: 6 and 16-21 or any one of SEQ ID NOs:34-59, 73-91, and 191-193 or any one of SEQ ID NOs:87-91 or SEQ ID NO:89] and includes the mutations C38S/C68G, C38S/C68A, C38S/C68V,C38S/C68D, C38S/C68E, or C38S/C68N relative to SEQ ID NO: 30.

In some embodiments, a subject IL-18 polypeptide includes an amino acidsequence having 90% or more sequence identity (e.g., 92% or more, 95% ormore, 97% or more, 98% or more, or 98.5% or more sequence identity) withhuman wild type IL-18 (SEQ ID NO: 30) [or any one of SEQ ID NOs: 6 and16-21 or any one of SEQ ID NOs: 34-59, 73-91, and 191-193 or any one ofSEQ ID NOs:87-91 or SEQ ID NO: 89] and includes the mutations C38S/C68S,C38S/C68G, C38S/C68A, C38S/C68D, or C38S/C68N relative to SEQ ID NO: 30.In some embodiments, a subject IL-18 polypeptide includes an amino acidsequence having 90% or more sequence identity (e.g., 92% or more, 95% ormore, 97% or more, 98% or more, or 98.5% or more sequence identity) withhuman wild type IL-18 (SEQ ID NO: 30) [or any one of SEQ ID NOs: 6 and16-21 or any one of SEQ ID NOs: 34-59, 73-91, and 191-193 or any one ofSEQ ID NOs:87-91 or SEQ ID NO: 89] and includes the mutations C38S/C68G,C38S/C68A, C38S/C68D, or C38S/C68N relative to SEQ ID NO: 30. In someembodiments, a subject IL-18 polypeptide includes an amino acid sequencehaving 90% or more sequence identity (e.g., 92% or more, 95% or more,97% or more, 98% or more, or 98.5% or more sequence identity) with humanwild type IL-18 (SEQ ID NO: 30) [or any one of SEQ ID NOs: 6 and 16-21or any one of SEQ ID NOs: 34-59, 73-91, and 191-193 or any one of SEQ IDNOs:87-91 or SEQ ID NO: 89] and includes the mutations C38S/C68Drelative to SEQ ID NO: 30. In some embodiments, a subject IL-18polypeptide includes an amino acid sequence having 90% or more sequenceidentity (e.g., 92% or more, 95% or more, 97% or more, 98% or more, or98.5% or more sequence identity) with human wild type IL-18 (SEQ ID NO:30) [or any one of SEQ ID NOs: 6 and 16-21 or any one of EQ ID NOs:34-59, 73-91, and 191-193 or any one of SEQ ID NOs:87-91 or SEQ ID NO:89] and includes the mutations C38S/C68G relative to SEQ ID NO: 30. Insome embodiments, a subject IL-18 polypeptide includes an amino acidsequence having 90% or more sequence identity (e.g., 92% or more, 95% ormore, 97% or more, 98% or more, or 98.5% or more sequence identity) withhuman wild type IL-18 (SEQ ID NO: 30) [or any one of SEQ ID NOs: 6 and16-21 or any one of SEQ ID NOs: 34-59, 73-91, and 191-193 or any one ofSEQ ID NOs:87-91 or SEQ ID NO: 89] and includes the mutations C38S/C68Arelative to SEQ ID NO: 30. In some embodiments, a subject IL-18polypeptide includes an amino acid sequence having 90% or more sequenceidentity (e.g., 92% or more, 95% or more, 97% or more, 98% or more, or98.5% or more sequence identity) with human wild type IL-18 (SEQ ID NO:30) [or any one of SEQ ID NOs: 6 and 16-21 or any one of SEQ ID NOs:34-59, 73-91, and 191-193 or any one of SEQ ID NOs:87-91 or SEQ ID NO:89] and includes the mutations C38S/C68N relative to SEQ ID NO: 30.

In some embodiments, the IL-18 variant polypeptide is a DR IL-18 variant(i.e., it binds to IL-18R and exhibits reduced binding to IL-18BP). Insome embodiments, the DR IL-18 variant binds to IL-18BP with a bindingaffinity that is 95% or less of the binding affinity of wild-type IL-18to IL-18BP (e.g., 90% or less, 80% or less, 70% or less, 60% or less,50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% orless, 2% or less, 1% or less, 0.05% or less, or 0.001% or less). In someembodiments, the DR IL-18 variant binds to IL-18BP with a bindingaffinity that is 10% or less (e.g., 5% or less, 2% or less, 1% or less,0.05% or less, or 0.001% or less) of the binding affinity of wild-typeIL-18 to IL-18BP.

In some embodiments, a subject DR IL-18 variant has a K_(D) for IL-18BPthat is 10 nM or greater (higher K_(D) means lower binding affinity). Insome embodiments, a subject DR-IL-18 variant polypeptide has a K_(D) forIL-18BP that is 20 nM or greater (e.g., 50 nM or greater, 100 nM orgreater, 500 nM or greater, or 1 μM or greater).

An IL-18 polypeptide (e.g., DR IL-18 polypeptide comprising mutations atpositions C38 and C68 relative to human wild type IL-18) can exhibitenhanced stability compared to an IL-18 polypeptide with a differentsequence (e.g., a wild type IL-18 sequence, or an IL-18 sequence thatlacks the C38 and C68 mutations but otherwise contains the same sequenceas the subject IL-18 polypeptide).

In some embodiments, stability of an IL-18 polypeptide (e.g., DR IL-18)disclosed herein can be evaluated by subjecting the polypeptide or aformulation disclosed herein that contains the polypeptide to freezethaw cycles, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 1, atleast 2, at least 3, at least 4, at least 5, at least 6, at least 7, atleast 8, at least 9, at least 10, at least 15, or at least 20 freezethat cycles. A freeze thaw cycle can comprise freezing the polypeptideor a formulation comprising the polypeptide (e.g., to about −20° C. or−80° C.) and thawing the polypeptide or a formulation (e.g., to about20° C., 23° C., 25° C., or 37° C.).

In some embodiments, stability of an IL-18 polypeptide (e.g., DR IL-18)disclosed herein can be evaluated by subjecting the polypeptide or aformulation disclosed herein that contains the polypeptide to agitation.Agitation can comprise, for example, incubating the polypeptide orformulation (e.g., at about 20° C., about 23° C., about 25° C., about30° C., about 37° C., or about 40° C.) with agitation at about 100 rpm,about 150 rpm, about 200 rpm, or about 250 rpm. Samples can be testedafter, for example, about 1 day, about 2 days, about 3 days, about 4days, about 5 days, about a week, or about 2 weeks of incubation at thetemperature and agitation rate.

Stability of an IL-18 polypeptide (e.g., DR IL-18) can be evaluated bycomparing the size of the main IL-18 peak measured by size exclusionchromatography before and after a stability assay disclosed herein(e.g., before and after freeze thaw cycles and/or agitation). In someembodiments, after exposure to the stability assay conditions, the mainpeak remains at least about 80%, at least about 85%, at least about 90%,at least about 91%, at least about 92%, at least about 93%, at leastabout 94%, at least about 95%, at least about 95.5%, at least about 96%,at least about 96.5%, at least about 97%, at least about 97.5%, at leastabout 98%, at least about 98.5%, at least about 99%, at least about99.1%, at least about 99.1%, at least about 99.3%, at least about 99.4%,at least about 99.5%, at least about 99.6%, at least about 99.7%, atleast about 99.8%, at least about 99.9%, at least about 99.95%, at leastabout 99.99%, or about 100% of the size of the main peak before exposureto the assay conditions.

In some embodiments, after exposure to the stability assay conditions,retention of the main peak at least about 1%, at least about 2%, atleast about 3%, at least about 4%, at least about 5%, at least about 6%,at least about 7%, at least about 8%, at least about 9%, at least about10%, at least about 11%, at least about 12%, at least about 13%, atleast about 14%, at least about 15%, at least about 20%, at least about25%, at least about 30%, at least about 40%, or at least about 50%greater than an IL-18 polypeptide with a different sequence (e.g., awild type IL-18 sequence, or an IL-18 sequence that lacks the C38 andC68 mutations but otherwise contains the same sequence as the subjectIL-18 polypeptide).

Stability of an IL-18 polypeptide (e.g., DR IL-18) can be evaluated bydetermining presence and size of one or more peaks measured by sizeexclusion chromatography that are indicative of formation of a dimer,multimer, aggregate, or degradation product, before and after astability assay disclosed herein (e.g., before and after freeze thawcycles and/or agitation). In some embodiments, after exposure of anIL-18 polypeptide (e.g., DR IL-18 in a formulation disclosed herein) tostability assay conditions, no or substantially no peak is detected thatis indicative of formation of dimers. In some embodiments, afterexposure of an IL-18 polypeptide (e.g., DR IL-18 in a formulationdisclosed herein) to stability assay conditions, no or substantially nopeak is detected that is indicative of formation of multimers. In someembodiments, after exposure of an IL-18 polypeptide (e.g., DR IL-18 in aformulation disclosed herein) to stability assay conditions, no orsubstantially no peak is detected that is indicative of formation ofaggregates. In some embodiments, after exposure of an IL-18 polypeptide(e.g., DR IL-18 in a formulation disclosed herein) to stability assayconditions, no or substantially no peak is detected that is indicativeof formation of degradation products.

In some embodiments, after exposure of an IL-18 polypeptide (e.g., DRIL-18 in a formulation disclosed herein) to stability assay conditions,the IL-18 polypeptide exhibits at least about 1%, at least about 2%, atleast about 3%, at least about 4%, at least about 5%, at least about 6%,at least about 7%, at least about 8%, at least about 9%, at least about10%, at least about 11%, at least about 12%, at least about 13%, atleast about 14%, at least about 15%, at least about 20%, at least about25%, at least about 30%, at least about 40%, at least about 50%, atleast about 60%, at least about 70%, at least about 80%, at least about90%, or at least about 95% less dimer, multimer, aggregate, and/ordegradation product formation compared to an IL-18 polypeptide with adifferent sequence (e.g., a wild type IL-18 sequence, or an IL-18sequence that lacks the C38 and C68 mutations but otherwise contains thesame sequence as the subject IL-18 polypeptide), e.g., as determined bysize exclusion chromatography.

In some embodiments, a DR IL-18 variant disclosed has a K_(D) forIL-18BP that is at least about 100 μM, at least about 10 μM, at leastabout 1 μM, at least about 900 nM, at least about 800 nM, at least about700 nM, at least about 600 nM, at least about 500 nM, at least about 400nM, at least about 300 nM, at least about 200 nM, at least about 150 nM,at least about 100 nM, at least about 90 nM, at least about 80 nM, atleast about 70 nM, at least about 60 nM, at least about 50 nM, at leastabout 40 nM, at least about 30 nM, at least about 20 nM, or at leastabout 10 nM. In some embodiments, a DR IL-18 variant disclosed does notbind IL-18BP, substantially does not bind IL-18BP, or binds IL-18BPbelow a limit of detection in an assay disclosed herein.

A DR IL-18 variant disclosed herein can exhibit binding to human IL-18R,for example, with can bind to human IL-18R at a comparable affinity towild type human IL-18, or can bind to human IL-18R with a higheraffinity than wild type human IL-18. In some embodiments, a DR IL-18variant disclosed herein binds to human IL-18R with at least comparableaffinity as wild type human IL-18 exhibits for human IL-18R.

In some embodiments, a DR IL-18 variant disclosed herein binds to humanIL-18Rα or human IL-18R with a K_(D) of, for example, less than about100 μM, less than about 10 μM, less than about 1 μM, less than about 900nM, less than about 800 nM, less than about 700 nM, less than about 600nM, less than about 500 nM, less than about 400 nM, less than about 300nM, less than about 200 nM, less than about 150 nM, less than about 100nM, less than about 90 nM, less than about 80 nM, less than about 70 nM,less than about 60 nM, less than about 50 nM, less than about 40 nM,less than about 30 nM, less than about 20 nM, less than about 10 nM,less than about 5 nM, less than about 4 nM, less than about 3 nM, lessthan about 2 nM, or less than about 1 nM.

In some embodiments, a DR IL-18 variant disclosed herein binds to humanIL-18Rα or human IL-18R with a K_(D) of, for example, about 1 pM toabout 1 μM, about 1 pM to about 500 nM, about 1 pM to about 400 nM,about 1 pM to about 300 nM, about 1 pM to about 200 nM, about 1 pM toabout 100 nM, about 1 pM to about 50 nM, about 100 pM to about 1 μM,about 100 pM to about 500 nM, about 100 pM to about 400 nM, about 100 pMto about 300 nM, about 100 pM to about 200 nM, about 100 pM to about 100nM, about 100 pM to about 50 nM, about 1 nM to about 1 μM, about 1 nM toabout 500 nM, about 1 nM to about 400 nM, about 1 nM to about 300 nM,about 1 nM to about 200 nM, about 1 nM to about 100 nM, about 1 nM toabout 75 nM, about 1 nM to about 50 nM, about 1 nM to about 40 nM, about1 nM to about 30 nM, or about 1 nM to about 10 nM.

In some embodiments, a DR IL-18 variant has an IL-18BP/IL-18Rdissociation constant ratio that is about at least 2-fold higher thanthe IL-18BP/IL-18R dissociation constant ratio of wild-type IL-18 (notethat an increased dissociation constant ratio implies a relativedecrease in IL-18BP binding relative to IL-18R binding). In someembodiments, DR IL-18 variant has an IL-18BP/IL-18R dissociationconstant ratio that is about at least 20-fold higher than theIL-18BP/IL-18R dissociation constant ratio of wild-type IL-18. In someembodiments, the DR IL-18 variant has an IL-18BP/IL-18R dissociationconstant ratio that is about at least 200-fold higher than theIL-18BP/IL-18R dissociation constant ratio of wild-type IL-18. In someembodiments, the DR IL-18 variant has an IL-18BP/IL-18R dissociationconstant ratio that is about at least 2,000-fold higher than theIL-18BP/IL-18R dissociation constant ratio of wild-type IL-18. In someembodiments, the DR IL-18 variant has an IL-18BP/IL-18R dissociationconstant ratio that is about at least 20,000-fold higher than theIL-18BP/IL-18R dissociation constant ratio of wild-type IL-18. In someembodiments, the DR IL-18 variant has an IL-18BP/IL-18R dissociationconstant ratio that is about at least 200,000-fold higher than theIL-18BP/IL-18R dissociation constant ratio of wild-type IL-18. In someembodiments, the DR IL-18 variant has an IL-18BP/IL-18R dissociationconstant ratio that is about at least 2,000,000-fold higher than theIL-18BP/IL-18R dissociation constant ratio of wild-type IL-18. In someembodiments, the DR IL-18 variant has an IL-18BP/IL-18R dissociationconstant ratio that is about at least 20,000,000-fold higher than theIL-18BP/IL-18R dissociation constant ratio of wild-type IL-18.

In some embodiments, the DR IL-18 variant has an IL-18BP/IL-18Rdissociation constant ratio that is about at least 3-fold higher thanthe IL-18BP/IL-18R dissociation constant ratio of wild-type IL-18. Insome embodiments, the DR IL-18 variant has an IL-18BP/IL-18Rdissociation constant ratio that is about at least 30-fold higher thanthe IL-18BP/IL-18R dissociation constant ratio of wild-type IL-18. Insome embodiments, the DR IL-18 variant has an IL-18BP/IL-18Rdissociation constant ratio that is about at least 300-fold higher thanthe IL-18BP/IL-18R dissociation constant ratio of wild-type IL-18. Insome embodiments, the DR IL-18 variant has an IL-18BP/IL-18Rdissociation constant ratio that is about at least 3,000-fold higherthan the IL-18BP/IL-18R dissociation constant ratio of wild-type IL-18.In some embodiments, the DR IL-18 variant has an IL-18BP/IL-18Rdissociation constant ratio that is about at least 30,000-fold higherthan the IL-18BP/IL-18R dissociation constant ratio of wild-type IL-18.In some embodiments, the DR IL-18 variant has an IL-18BP/IL-18Rdissociation constant ratio that is about at least 300,000-fold higherthan the IL-18BP/IL-18R dissociation constant ratio of wild-type IL-18.In some embodiments, the DR IL-18 variant has an IL-18BP/IL-18Rdissociation constant ratio that is about at least 3,000,000-fold higherthan the IL-18BP/IL-18R dissociation constant ratio of wild-type IL-18.In some embodiments, the DR IL-18 variant has an IL-18BP/IL-18Rdissociation constant ratio that is about at least 30,000,000-foldhigher than the IL-18BP/IL-18R dissociation constant ratio of wild-typeIL-18.

In some embodiments, the DR IL-18 variant has an IL-18BP/IL-18Rdissociation constant ratio that is about at least 10-fold higher thanthe IL-18BP/IL-18R dissociation constant ratio of wild-type IL-18. Insome embodiments, the DR IL-18 variant has an IL-18BP/IL-18Rdissociation constant ratio that is about at least 100-fold higher thanthe IL-18BP/IL-18R dissociation constant ratio of wild-type IL-18. Insome embodiments, the DR IL-18 variant has an IL-18BP/IL-18Rdissociation constant ratio that is about at least 1000-fold higher thanthe IL-18BP/IL-18R dissociation constant ratio of wild-type IL-18. Insome embodiments, the DR IL-18 variant has an IL-18BP/IL-18Rdissociation constant ratio that is about at least 10,000-fold higherthan the IL-18BP/IL-18R dissociation constant ratio of wild-type IL-18.In some embodiments, the DR IL-18 variant has an IL-18BP/IL-18Rdissociation constant ratio that is about at least 100,000-fold higherthan the IL-18BP/IL-18R dissociation constant ratio of wild-type IL-18.In some embodiments, the DR IL-18 variant has an IL-18BP/IL-18Rdissociation constant ratio that is about at least 1,000,000-fold higherthan the IL-18BP/IL-18R dissociation constant ratio of wild-type IL-18.In some embodiments, the DR IL-18 variant has an IL-18BP/IL-18Rdissociation constant ratio that is about at least 10,000,000-foldhigher than the IL-18BP/IL-18R dissociation constant ratio of wild-typeIL-18. In some embodiments, the DR IL-18 variant has an IL-18BP/IL-18Rdissociation constant ratio that is about at least 100,000,000-foldhigher than the IL-18BP/IL-18R dissociation constant ratio of wild-typeIL-18.

In some embodiments, a subject DR IL-18 variant has an inhibitorconstant (Ki) for IL-18BP that is greater than 3 nM (e.g., 5 nM or more,10 nM or more, 50 nM or more, 100 nM or more, 500 nM or more, 750 nM ormore, or 1 μM or more). In some embodiments, a subject DR-IL-18 variantpolypeptide has a Ki for IL-18BP that is 500 nM or more. In someembodiments, a subject DR-IL-18 variant polypeptide has a Ki for IL-18BPthat is 1 μM or more.

In some embodiments, a subject IL-18 variant polypeptide (a DR-IL-18)that binds to IL-1518R and exhibits reduced binding to IL-18BP has a Kifor IL-18BP that is greater than 200 nM (e.g., 500 nM or more, 750 nM ormore, or 1 μM or more). In some embodiments, a subject DR-IL-18 variantpolypeptide has a Ki for IL-18BP that is 1 μM or more.

In some embodiments, a subject IL-18 variant polypeptide (a DR-IL-18)that binds to IL-18R and exhibits substantially reduced binding toIL-18BP has an inhibitor constant (Ki) for IL-18BP that is at least2-fold higher than the Ki of wild type IL-18 for IL-18BP (i.e., the Kiof the subject IL-18 variant polypeptide for IL-18BP is at least 2-foldrelative to the Ki of WT IL-18 for IL-18BP). For example, in some casesa subject DR-IL-18 variant polypeptide has a Ki for IL-18BP that is atleast 5-fold higher (e.g., at least 10-fold, at least 50-fold, at least100-fold, at least 200-fold, at least 500-fold, or at least 1000-folderhigher) than the Ki of wild type IL-18 for IL-18BP.

In some embodiments, a subject IL-18 variant polypeptide (a DR-IL-18)that binds to IL-18R and exhibits substantially reduced binding toIL-18BP has an EC50 for IL-18BP that is at least 2-fold higher than theEC50 of wild type IL-18 for IL-18BP (i.e., the EC50 of the subject IL-18variant polypeptide for IL-18BP is at least 2-fold relative to the EC50of WT IL-18 for IL-18BP). For example, in some cases a subject DR-IL-18variant polypeptide has a EC50 for IL-18BP that is at least 5-foldhigher (e.g., at least 10-fold, at least 50-fold, at least 100-fold, atleast 200-fold, at least 500-fold, or at least 1000-folder higher) thanthe EC50 of wild type IL-18 for IL-18BP.

In any of the above cases, the DR IL-18 variant can include C38/C68mutations in any of the combinations presented above (e.g. in some casesC38S/C68S, C38S/C68G, C38S/C68A, C38S/C68A, C38S/C68V, C38S/C68D,C38S/C68E, or C38S/C68N; in some cases C38S/C68S, C38S/C68G, C38S/C68A,C38S/C68D, or C38S/C68N; in some cases C38S/C68G, C38S/C68A, C38S/C68D,or C38S/C68N; C38S/C68D; in some cases C38S/C68G; in some casesC38S/C68A; in some cases C38S/C68N; in some cases C38S/C68S).

Likewise, for any of the below, the DR IL-18 variant can include C38/C68mutations in any of the combinations presented above (e.g. in some casesC38S/C68S, C38S/C68G, C38S/C68A, C38S/C68V, C38S/C68D, C38S/C68E, orC38S/C68N; in some cases C38S/C68G, C38S/C68A, C38S/C68V, C38S/C68D,C38S/C68E, or C38S/C68N; in some cases C38S/C68S, C38S/C68G, C38S/C68A,C38S/C68D, or C38S/C68N; in some cases C38S/C68G, C38S/C68A, C38S/C68D,or C38S/C68N; C38S/C68D; in some cases C38S/C68G; in some casesC38S/C68A; in some cases C38S/C68N; in some cases C38S/C68S).

In some embodiments a DR-IL-18 variant comprises at least one mutation(e.g., at least 2, at least 3, at least 4, at least 5, or at least 6mutations) at an amino acid position selected from the group consistingof Y1, L5, K8, M51, K53, S55, Q56, P57, G59, M60, E77, Q103, S105, D110,N111, M113, V153, and N155. In various embodiments, the human IL-18variant polypeptide comprises at least 4 mutations at amino acidpositions selected from the group consisting of Y1, L5, K8, M51, K53,S55, Q56, P57, G59, M60, E77, Q103, S105, D110, N111, M113, V153, andN155. In various embodiments, the human IL-18 variant polypeptidecomprises at least 6 mutations at amino acid positions selected from thegroup consisting of Y1, L5, K8, M51, K53, S55, Q56, P57, G59, M60, E77,Q103, S105, D110, N111, M113, V153, and N155. In various embodiments,the human IL-18 variant polypeptide comprises at least one mutation(e.g., at least 2, at least 3, at least 4, at least 5, or at least 6mutations) at an amino acid position selected from the group consistingof Y1, L5, K8, S55, Q56, P57, G59, E77, Q103, S105, D110, N111, M113,V153, and N155. In some embodiments, a human IL-18 variant polypeptidecomprises at least one mutation (e.g., at least 2, at least 3, at least4, at least 5, or at least 6 mutations) at an amino acid positionselected from the group consisting of Y1H, Y1R, L5H, L5I, L5Y, K8Q, K8R,M51T, M51K, M51D, M51N, M51E, M51R, K53R, K53G, K53S, K53T, S55K, S55R,Q56E, Q56A, Q56R, Q56V, Q56G, Q56K, Q56L, P57L, P57G, P57A, P57K, G59T,G59A, M60K, M60Q, M60R, M60L, E77D, Q103E, Q103K, Q103P, Q103A, Q103R,S105R, S105D, S105K, S105N, S105A, D110H, D110K, D110N, D110Q, D110E,D110S, D110G, N111H, N111Y, N111D, N111R, N111S, N111G, M113V, M113R,M113T, M113K, V153I, V153T, V153A, N155K, and N155H.

In some embodiments, a DR IL-18 variant polypeptide comprises the aminoacid sequence set forth in any one of SEQ ID NOs: 34-59, 73-91, and191-193. In some embodiments, a DR IL-18 variant polypeptide comprisesan amino acid sequence having 80% or more sequence identity (e.g., 85%or more, 90% or more, 95% or more, 98% or more, 99% or more, or 100%)with the amino acid sequence set forth in any one of SEQ ID NOs: 34-59,73-91, and 191-193. In some embodiments, a DR IL-18 variant polypeptidecomprises an amino acid sequence having 85% or more sequence identity(e.g., 90% or more, 95% or more, 98% or more, 99% or more, or 100%) withthe amino acid sequence set forth in any one of SEQ ID NOs: 34-59,73-91, and 191-193. In some embodiments, a DR IL-18 variant polypeptidecomprises an amino acid sequence having 90% or more sequence identity(e.g., 95% or more, 98% or more, 99% or more, or 100%) with the aminoacid sequence set forth in any one of SEQ ID NOs: 34-59, 73-91, and191-193.

In some cases a subject DR-IL-18 variant includes at least one mutation(e.g., at least 2, at least 3, or at least 4 mutations) at an amino acidposition selected from the group consisting of M51, M60, S105, D110, andN111, relative to SEQ ID NO: 30. In some cases a subject DR-IL-18variant includes at least 3 mutations at amino acid positions selectedfrom the group consisting of M51, M60, S105, D110, and N111, relative toSEQ ID NO: 30. In some cases a subject DR-IL-18 variant includes atleast one mutation (e.g., at least 2, at least 3, or at least 4mutations) at an amino acid position selected from the group consistingof M51, M60, S105, D110, and N111, relative to SEQ ID NO: 30, where M51is T, K, D, E, R, or N; M60 is K, Q, L, or R; S105 is R, D, K, A, or N;D110 is H, K, N, Q, E, N, S, or G; and N111 is H, D, Y, R, S, or G. Insome cases a subject DR-IL-18 variant includes at least 3 mutations(e.g., at least 2, at least 3, or at least 4 mutations) at amino acidpositions selected from the group consisting of M51, M60, S105, D110,and N111, relative to SEQ ID NO: 30, where M51 is T, K, D, E, R, or N;M60 is K, Q, L, or R; S105 is R, D, K, A, or N; D110 is H, K, N, Q, E,N, S, or G; and N111 is H, D, Y, R, S, or G. In some cases, a subjectDR-IL-18 variant includes at least one mutation (e.g., at least 2, atleast 3, or at least 4 mutations) at an amino acid position selectedfrom the group consisting of M51, M60, S105, D110, and N111, relative toSEQ ID NO: 30, where M51 is T or K; M60 is K or L; S105 is D, N, or A;D110 is K, N, S, or G; and N111 is H, Y, G, or R.

In some cases a subject DR-IL-18 variant includes the mutations at aminoacid positions M51, M60, S105, D110, and N111, relative to SEQ ID NO:30. For example, in some cases a subject DR-IL-18 variant includes themutations M51, M60, S105, D110, and N111, relative to SEQ ID NO: 30,where M51 is T, K, D, E, R, or N; M60 is K, Q, L, or R; S105 is R, D, K,A, or N; D110 is H, K, N, Q, E, N, S, or G; and N111 is H, D, Y, R, S,or G. In some cases, a subject DR-IL-18 variant includes the mutationsM51, M60, S105, D110, and N111, relative to SEQ ID NO: 30, where M51 isT or K; M60 is K or L; S105 is D, N, or A; D110 is K, N, S, or G; andN111 is H, Y, G, or R. In other words, in some cases a subject DR-IL-18variant includes the mutations {M51T or M51K}; {M60K or M60L}; {S105D,S105N, S105A}; {D110K, D110N, D110S, or D110G}; and {N111H, N111Y,N111R, or N111G}, relative to SEQ ID NO: 30.

In some cases a subject DR-IL-18 variant includes at least one mutation(e.g., at least 2, at least 3, or at least 4 mutations) at an amino acidposition selected from the group consisting of M51, K53, Q56, S105, andN111, relative to SEQ ID NO: 30. In some cases a subject DR-IL-18variant includes at least 3 mutations at amino acid positions selectedfrom the group consisting of M51, K53, Q56, S105, and N111, relative toSEQ ID NO: 30. In some cases a subject DR-IL-18 variant includes atleast one mutation (e.g., at least 2, at least 3, or at least 4mutations) selected from the group consisting of M51, K53, Q56, S105,and N111, relative to SEQ ID NO: 30, where M51 is E, R, or K; K53 is G,S, or T; Q56 is E, A, R, V, G, K, or L; S105 is N, S, K, or G; and N111is R, S, G, or D. In some cases, a subject DR-IL-18 variant includes atleast 3 mutations selected from the group consisting of M51, K53, Q56,S105, and N111, relative to SEQ ID NO: 30, where M51 is E, R, or K; K53is G, S, or T; Q56 is E, A, R, V, G, K, or L; S105 is N, S, K, or G; andN111 is R, S, G, or D. In some cases, a subject DR-IL-18 variantincludes at least one mutation (e.g., at least 2, at least 3, or atleast 4 mutations) selected from the group consisting of M51, K53, Q56,S105, and N111, relative to SEQ ID NO: 30, where M51 is K; K53 is G orS; Q56 is G, R, or L; S105 is S, N, or G; and N111 is G or R.

In some cases a subject DR-IL-18 variant includes mutations at aminoacid positions M51, K53, Q56, D110, and N111, relative to SEQ ID NO: 30.For example, in some cases a subject DR-IL-18 variant includes themutations M51, K53, Q56, D110, and N111, relative to SEQ ID NO: 30,where M51 is E, R, or K; K53 is G, S, or T; Q56 is E, A, R, V, G, K, orL; D110 is S, N, G, or K; and N111 is R, S, G, or D. In some cases, asubject DR-IL-18 variant includes the mutations M51, K53, Q56, D110, andN111, relative to SEQ ID NO: 30, where M51 is K; K53 is G or S; Q56 isG, R, or L; D110 is S, N, or G; and N111 is G or R. In other words, insome cases a subject DR-IL-18 variant includes the mutations {M51K};{K53G or K535}; {Q56G, Q56R, or Q56L}; {D110S, D110N, or D110G}; and{N111R, or N111G}, relative to SEQ ID NO: 30.

In some cases, in addition to the mutations of the previous paragraphthe DR IL-18 variant also includes mutations at positions P57 and M60,and in some cases S105 as well. For example, in some cases P57 is A, K,G or L; M60 is L or R—and in some such case S105 is A, N, or D. In somecases, P57 is A (i.e., P57A) and M60 is L (i.e., M60L)—and in some suchcase S105 is A, N, or D (in some cases S105D).

In some cases a subject DR-IL-18 variant comprises an amino acidsequence having 85% or more sequence identity (e.g., 90% or more, 92% ormore, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more,98% or more, or 99% or more sequence identity) with the wild type humanIL-18 amino acid sequence set forth as SEQ ID NO: 30. As such in somecases a subject DR-IL-18 variant comprises an amino acid sequence that(i) has 85% or more sequence identity (e.g., 90% or more, 92% or more,93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% ormore, or 99% or more sequence identity) with the wild type human IL-18amino acid sequence set forth as SEQ ID NO: 30; and (ii) includes atleast one mutation (e.g., at least 2, at least 3, at least 4, at least5, or at least 6 mutations) relative to wild type IL-18 (e.g., humanIL-18) (e.g., see all of the mutation combinations above).

In some cases, a subject DR-IL-18 variant comprises an amino acidsequence having 85% or more sequence identity (e.g., 90% or more, 92% ormore, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more,98% or more, or 99% or more sequence identity) with the amino acidsequence set forth in any one of SEQ ID NOs: 34-59, 73-91, and 191-193.As such in some cases a subject DR-IL-18 variant comprises an amino acidsequence that (i) has 85% or more sequence identity (e.g., 90% or more,92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% ormore, 98% or more, or 99% or more sequence identity) with the amino acidsequence set forth in any one of SEQ ID NOs: 34-59, 73-91, and 191-193;and (ii) includes at least one mutation (e.g., at least 2, at least 3,at least 4, at least 5, or at least 6 mutations) relative to wild typeIL-18 (e.g., human IL-18).

In some cases, a subject DR-IL-18 variant comprises an amino acidsequence that (i) has 85% or more sequence identity (e.g., 90% or more,92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% ormore, 98% or more, or 99% or more sequence identity) with the wild typehuman IL-18 amino acid sequence set forth as SEQ ID NO: 30; and (ii)includes at least one mutation (e.g., at least 2, at least 3, at least4, at least 5, or at least 6 mutations) at amino acid positions selectedfrom the group consisting of Y1, L5, K8, M51, K53, S55, Q56, P57, G59,M60, E77, Q103, S105, D110, N111, M113, V153, and N155, relative to SEQID NO: 30. In some cases a subject DR-IL-18 variant comprises an aminoacid sequence that (i) has 85% or more sequence identity (e.g., 90% ormore, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more,97% or more, 98% or more, or 99% or more sequence identity) with thewild type human IL-18 amino acid sequence set forth as SEQ ID NO: 30;and (ii) includes at least 4 mutations at amino acid positions selectedfrom the group consisting of Y1, L5, K8, M51, K53, S55, Q56, P57, G59,M60, E77, Q103, S105, D110, N111, M113, V153, and N155, relative to SEQID NO: 30. In some cases a subject DR-IL-18 variant comprises an aminoacid sequence that (i) has 85% or more sequence identity (e.g., 90% ormore, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more,97% or more, 98% or more, or 99% or more sequence identity) with thewild type human IL-18 amino acid sequence set forth as SEQ ID NO: 30;and (ii) includes at least 6 mutations at amino acid positions selectedfrom the group consisting of Y1, L5, K8, M51, K53, S55, Q56, P57, G59,M60, E77, Q103, S105, D110, N111, M113, V153, and N155, relative to SEQID NO: 30. In some cases a subject DR-IL-18 variant comprises an aminoacid sequence that (i) has 85% or more sequence identity (e.g., 90% ormore, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more,97% or more, 98% or more, or 99% or more sequence identity) with thewild type human IL-18 amino acid sequence set forth as SEQ ID NO: 30;and (ii) includes at least one mutation (e.g., at least 2, at least 3,at least 4, at least 5, or at least 6 mutations) at an amino acidposition selected from the group consisting of Y1, L5, K8, S55, Q56,P57, G59, E77, Q103, S105, D110, N111, M113, V153, and N155, relative toSEQ ID NO: 30.

In some cases a subject DR-IL-18 variant comprises an amino acidsequence that (i) has 85% or more sequence identity (e.g., 90% or more,92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% ormore, 98% or more, or 99% or more sequence identity) with the wild typehuman IL-18 amino acid sequence set forth as SEQ ID NO: 30; and (ii)includes at least one mutation (e.g., at least 2, at least 3, or atleast 4 mutations) at an amino acid position selected from the groupconsisting of M51, M60, S105, D110, and N111, relative to SEQ ID NO: 30.In some cases a subject DR-IL-18 variant comprises an amino acidsequence that (i) has 85% or more sequence identity (e.g., 90% or more,92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% ormore, 98% or more, or 99% or more sequence identity) with the wild typehuman IL-18 amino acid sequence set forth as SEQ ID NO: 30; and (ii)includes at least 3 mutations at amino acid positions selected from thegroup consisting of M51, M60, S105, D110, and N111, relative to SEQ IDNO: 30. In some cases a subject DR-IL-18 variant comprises an amino acidsequence that (i) has 85% or more sequence identity (e.g., 90% or more,92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% ormore, 98% or more, or 99% or more sequence identity) with the wild typehuman IL-18 amino acid sequence set forth as SEQ ID NO: 30; and (ii)includes at least one mutation (e.g., at least 2, at least 3, or atleast 4 mutations) selected from the group consisting of M51, M60, S105,D110, and N111, relative to SEQ ID NO: 30, where M51 is T, K, D, E, R,or N; M60 is K, Q, L, or R; S105 is R, D, K, A, or N; D110 is H, K, N,Q, E, N, S, or G; and N111 is H, D, Y, R, S, or G. In some cases asubject DR-IL-18 variant comprises an amino acid sequence that (i) has85% or more sequence identity (e.g., 90% or more, 92% or more, 93% ormore, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more,or 99% or more sequence identity) with the wild type human IL-18 aminoacid sequence set forth as SEQ ID NO: 30; and (ii) includes at least 3mutations at amino acid positions selected from the group consisting ofM51, M60, S105, D110, and N111, relative to SEQ ID NO: 30, where M51 isT, K, D, E, R, or N; M60 is K, Q, L, or R; S105 is R, D, K, A, or N;D110 is H, K, N, Q, E, N, S, or G; and N111 is H, D, Y, R, S, or G. Insome cases a subject DR-IL-18 variant comprises an amino acid sequencethat (i) has 85% or more sequence identity (e.g., 90% or more, 92% ormore, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more,98% or more, or 99% or more sequence identity) with the wild type humanIL-18 amino acid sequence set forth as SEQ ID NO: 30; and (ii) includesat least one mutation (e.g., at least 2, at least 3, or at least 4mutations) at an amino acid position selected from the group consistingof M51, M60, S105, D110, and N111, relative to SEQ ID NO: 30, where M51is T or K; M60 is K or L; S105 is D, N, or A; D110 is K, N, S, or G; andN111 is H, Y, G, or R.

In some cases a subject DR-IL-18 variant comprises an amino acidsequence that (i) has 85% or more sequence identity (e.g., 90% or more,92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% ormore, 98% or more, or 99% or more sequence identity) with the wild typehuman IL-18 amino acid sequence set forth as SEQ ID NO: 30; and (ii)includes the mutations at positions M51, M60, S105, D110, and N111,relative to SEQ ID NO: 30. In some cases a subject DR-IL-18 variantcomprises an amino acid sequence that (i) has 85% or more sequenceidentity (e.g., 90% or more, 92% or more, 93% or more, 94% or more, 95%or more, 96% or more, 97% or more, 98% or more, or 99% or more sequenceidentity) with the wild type human IL-18 amino acid sequence set forthas SEQ ID NO: 30; and (ii) includes the mutations M51, M60, S105, D110,and N111, relative to SEQ ID NO: 30, where M51 is T, K, D, E, R, or N;M60 is K, Q, L, or R; S105 is R, D, K, A, or N; D110 is H, K, N, Q, E,N, S, or G; and N111 is H, D, Y, R, S, or G.

In some cases a subject DR-IL-18 variant comprises an amino acidsequence that (i) has 85% or more sequence identity (e.g., 90% or more,92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% ormore, 98% or more, or 99% or more sequence identity) with the wild typehuman IL-18 amino acid sequence set forth as SEQ ID NO: 30; and (ii)includes the mutations M51, M60, S105, D110, and N111, relative to SEQID NO: 30, where M51 is T or K; M60 is K or L; S105 is D, N, or A; D110is K, N, S, or G; and N111 is H, Y, G, or R.

In some cases a subject DR-IL-18 variant comprises an amino acidsequence that (i) has 85% or more sequence identity (e.g., 90% or more,92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% ormore, 98% or more, or 99% or more sequence identity) with the wild typehuman IL-18 amino acid sequence set forth as SEQ ID NO: 30; and (ii)includes at least one mutation (e.g., at least 2, at least 3, or atleast 4 mutations) at positions selected from the group consisting ofM51, K53, Q56, D110, and N111, relative to SEQ ID NO: 30. In some casesa subject DR-IL-18 variant comprises an amino acid sequence that (i) has85% or more sequence identity (e.g., 90% or more, 92% or more, 93% ormore, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more,or 99% or more sequence identity) with the wild type human IL-18 aminoacid sequence set forth as SEQ ID NO: 30; and (ii) includes at least 3mutations at positions selected from the group consisting of M51, K53,Q56, D110, and N111, relative to SEQ ID NO: 30. In some cases a subjectDR-IL-18 variant comprises an amino acid sequence that (i) has 85% ormore sequence identity (e.g., 90% or more, 92% or more, 93% or more, 94%or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% ormore sequence identity) with the wild type human IL-18 amino acidsequence set forth as SEQ ID NO: 30; and (ii) includes at least onemutation (e.g., at least 2, at least 3, or at least 4 mutations)selected from the group consisting of M51, K53, Q56, D110, and N111,relative to SEQ ID NO: 30, where M51 is E, R, or K; K53 is G, S, or T;Q56 is E, A, R, V, G, K, or L; D110 is N, S, K, or G; and N111 is R, S,G, or D. In some cases a subject DR-IL-18 variant comprises an aminoacid sequence that (i) has 85% or more sequence identity (e.g., 90% ormore, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more,97% or more, 98% or more, or 99% or more sequence identity) with thewild type human IL-18 amino acid sequence set forth as SEQ ID NO: 30;and (ii) includes at least 3 mutations selected from the groupconsisting of M51, K53, Q56, D110, and N111, relative to SEQ ID NO: 30,where M51 is E, R, or K; K53 is G, S, or T; Q56 is E, A, R, V, G, K, orL; D110 is N, S, K, or G; and N111 is R, S, G, or D. In some cases asubject DR-IL-18 variant comprises an amino acid sequence that (i) has85% or more sequence identity (e.g., 90% or more, 92% or more, 93% ormore, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more,or 99% or more sequence identity) with the wild type human IL-18 aminoacid sequence set forth as SEQ ID NO: 30; and (ii) includes at least onemutation (e.g., at least 2, at least 3, or at least 4 mutations)selected from the group consisting of M51, K53, Q56, D110, and N111,relative to SEQ ID NO: 30, where M51 is K; K53 is G or S; Q56 is G, R,or L; D110 is S, N, or G; and N111 is G or R.

In some cases a subject DR-IL-18 variant comprises an amino acidsequence that (i) has 85% or more sequence identity (e.g., 90% or more,92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% ormore, 98% or more, or 99% or more sequence identity) with the wild typehuman IL-18 amino acid sequence set forth as SEQ ID NO: 30; and (ii)includes mutations at positions M51, K53, Q56, D110, and N111, relativeto SEQ ID NO: 30. In some cases a subject DR-IL-18 variant comprises anamino acid sequence that (i) has 85% or more sequence identity (e.g.,90% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% ormore, 97% or more, 98% or more, or 99% or more sequence identity) withthe wild type human IL-18 amino acid sequence set forth as SEQ ID NO:30; and (ii) includes the mutations M51, K53, Q56, D110, and N111,relative to SEQ ID NO: 30, where M51 is E, R, or K; K53 is G, S, or T;Q56 is E, A, R, V, G, K, or L; D110 is N, S, K, or G; and N111 is R, S,G, or D. In some cases a subject DR-IL-18 variant comprises an aminoacid sequence that (i) has 85% or more sequence identity (e.g., 90% ormore, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more,97% or more, 98% or more, or 99% or more sequence identity) with thewild type human IL-18 amino acid sequence set forth as SEQ ID NO: 30;and (ii) includes the mutations M51, K53, Q56, D110, and N111, relativeto SEQ ID NO: 30, where M51 is K; K53 is G or S; Q56 is G, R, or L; D110is S, N, or G; and N111 is G or R.

In some cases, in addition to the mutations of the previous paragraphthe DR IL-18 variant also includes mutations at positions P57 and M60,and in some cases S105 as well. For example, in some cases P57 is A, K,G or L; M60 is L or R—and in some such case S105 is A, N, or D. In somecases, P57 is A (i.e., P57A) and M60 is L (i.e., M60L)—and in some suchcase S105 is A, N, or D (in some cases S105D).

Examples of murine IL-18 variant polypeptides (in this case DR IL-18variants) include, but are not limited to mCS1 (SEQ ID NO: 60), mCS2(SEQ ID NO: 61), mC1 (SEQ ID NO: 62), mA12 (SEQ ID NO: 63), mE8 (SEQ IDNO: 64), mC10 (SEQ ID NO: 65), mB7 (SEQ ID NO: 66), mB1 (SEQ ID NO: 67),mD1 (SEQ ID NO: 68), mH7 (SEQ ID NO: 69), mA7 (SEQ ID NO: 70), mE1 (SEQID NO: 71), and mH3 (SEQ ID NO: 72).

As noted above, any of the IL-18 variants herein can be ‘stabilized’ byfurther mutating C38 and C68 relative to SEQ ID NO: 30. As anillustrative example, in some cases, a subject stabilized DR-IL-18variant comprises an amino acid sequence that (i) has 85% or moresequence identity (e.g., 90% or more, 92% or more, 93% or more, 94% ormore, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or moresequence identity) with the wild type human IL-18 amino acid sequenceset forth as SEQ ID NO: 30; (ii) includes mutations at positions M51,K53, Q56, D110, and N111, relative to SEQ ID NO: 30 [or any one of SEQID NOs: 6 and 16-21 or any one of SEQ ID NOs: 34-59, 73-91, and 191-193or any one of SEQ ID NOs: 87-91 or SEQ ID NO: 89]; and (iii) includesmutations at C38 and C68 (e.g., C38S/C68S, C38S/C68G, C38S/C68A,C38S/C68V, C38S/C68D, C38S/C68E, or C38S/C68N—in some cases C38S/C68G,C38S/C68A, C38S/C68V, C38S/C68D, C38S/C68E, or C38S/C68N—in some casesC38S/C68S, C38S/C68G, C38S/C68A, C38S/C68D, or C38S/C68N—in some casesC38S/C68G, C38S/C68A, C38S/C68D, or C38S/C68N—in some cases C38S/C68D—insome cases C38S/C68G—in some cases C38S/C68A—in some cases C38S/C68N).In some cases a subject DR-IL-18 variant comprises an amino acidsequence that (i) has 85% or more sequence identity (e.g., 90% or more,92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% ormore, 98% or more, or 99% or more sequence identity) with the wild typehuman IL-18 amino acid sequence set forth as SEQ ID NO: 30 [or any oneof SEQ ID NOs: 6 and 16-21 or any one of SEQ ID NOs: 34-59, 73-91, and191-193 or any one of SEQ ID NOs:87-91 or SEQ ID NO: 89]; (ii) includesthe mutations M51, K53, Q56, D110, and N111, relative to SEQ ID NO: 30,where M51 is E, R, or K; K53 is G, S, or T; Q56 is E, A, R, V, G, K, orL; D110 is N, S, K, or G; and N111 is R, S, G, or D; and (iii) includesmutations at C38 and C68 (e.g., C38S/C68S, C38S/C68G, C38S/C68A,C38S/C68V, C38S/C68D, C38S/C68E, or C38S/C68N—in some cases C38S/C68G,C38S/C68A, C38S/C68V, C38S/C68D, C38S/C68E, or C38S/C68N—in some casesC38S/C68S, C38S/C68G, C38S/C68A, C38S/C68D, or C38S/C68N—in some casesC38S/C68G, C38S/C68A, C38S/C68D, or C38S/C68N—in some cases C38S/C68D—insome cases C38S/C68G—in some cases C38S/C68A—in some cases C38S/C68N).In some cases a subject DR-IL-18 variant comprises an amino acidsequence that (i) has 85% or more sequence identity (e.g., 90% or more,92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% ormore, 98% or more, or 99% or more sequence identity) with the wild typehuman IL-18 amino acid sequence set forth as SEQ ID NO: 30 [or any oneof SEQ ID NOs: 6 and 16-21 or any one of SEQ ID NOs: 34-59, 73-91, and191-193 or any one of SEQ ID NOs:87-91 or SEQ ID NO: 89]; (ii) includesthe mutations M51, K53, Q56, D110, and N111, relative to SEQ ID NO: 30,where M51 is K; K53 is G or S; Q56 is G, R, or L; D110 is S, N, or G;and N111 is G or R; and (iii) includes mutations at C38 and C68 (e.g.,C38S/C68S, C38S/C68G, C38S/C68A, 8S/C68V, C38S/C68D, C38S/C68E, orC38S/C68N—in some cases C38S/C68G, C38S/C68A, C38S/C68V, C38S/C68D,C38S/C68E, or C38S/C68N—in some cases C38S/C68S, C38S/C68G, C38S/C68A,C38S/C68D, or C38S/C68N—in some cases C38S/C68G, C38S/C68A, C38S/C68D,or C38S/C68N—in some cases C38S/C68D—in some cases C38S/C68G—in somecases C38S/C68A—in some cases C38S/C68N).

In some cases, in addition to the mutations of the previous paragraphthe DR IL-18 variant also includes mutations at positions P57 and M60,and in some cases S105 as well. For example, in some cases P57 is A, K,G or L; M60 is L or R—and in some such case S105 is A, N, or D. In somecases, P57 is A (i.e., P57A) and M60 is L (i.e., M60L)—and in some suchcase S105 is A, N, or D (in some cases S105D).

In some cases a subject DR IL-18 variant (i) includes mutationsC38S/C68S, C38S/C68G, C38S/C68A, C38S/C68V, C38S/C68D, C38S/C68E, orC38S/C68N (relative to SEQ ID NO: 30); (ii) has 90% or more sequenceidentity (e.g., 92% or more, 93% or more, 94% or more, 95% or more, 96%or more, 97% or more, 98% or more, or 99% or more sequence identity)with the wild type human IL-18 amino acid sequence set forth as SEQ IDNO: 30; and (iii) includes mutations as positions M51, K53, Q56, D110,and N111 relative to SEQ ID NO: 30. In some such cases the variantfurther includes mutations at positions P57 and M60. In some such casesthe variant further includes a mutation at position S105.

In some cases a subject DR IL-18 variant (i) includes mutationsC38S/C68S, C38S/C68G, C38S/C68A, C38S/C68V, C38S/C68D, C38S/C68E, orC38S/C68N (relative to SEQ ID NO: 30); (ii) has 90% or more sequenceidentity (e.g., 92% or more, 93% or more, 94% or more, 95% or more, 96%or more, 97% or more, 98% or more, or 99% or more sequence identity)with the wild type human IL-18 amino acid sequence set forth as SEQ IDNO: 30; and (iii) includes mutations as positions M51, K53, Q56, D110,and N111 relative to SEQ ID NO: 30, where M51 is K; K53 is G or S; Q56is G, R, or L; D110 is S, N, or G; and N111 is G or R. In some suchcases the variant further includes mutations at positions where P57 isA, K, G or L; and M60 is L or R. In some such cases the variant furtherincludes a mutation at position S105 where S105 is A, N, or D.

In some cases a subject DR IL-18 variant (i) includes mutationsC38S/C68G, 20 C38S/C68A, C38S/C68V, C38S/C68D, C38S/C68E, or C38S/C68N(relative to SEQ ID NO: 30); (ii) has 90% or more sequence identity(e.g., 92% or more, 93% or more, 94% or more, 95% or more, 96% or more,97% or more, 98% or more, or 99% or more sequence identity) with thewild type human IL-18 amino acid sequence set forth as SEQ ID NO: 30;and (iii) includes mutations as positions M51, K53, Q56, D110, and N111relative to SEQ ID NO: 30. In some such cases the variant furtherincludes mutations at positions P57 and M60. In some such cases thevariant further includes a mutation at position S105.

In some cases a subject DR IL-18 variant (i) includes mutationsC38S/C68G, C38S/C68A, C38S/C68V, C38S/C68D, C38S/C68E, or C38S/C68N(relative to SEQ ID NO: 30); (ii) has 90% or more sequence identity(e.g., 92% or more, 93% or more, 94% or more, 95% or more, 96% or more,97% or more, 98% or more, or 99% or more sequence identity) with thewild type human IL-18 amino acid sequence set forth as SEQ ID NO: 30;and (iii) includes mutations as positions M51, K53, Q56, D110, and N111relative to SEQ ID NO: 30, where M51 is K; K53 is G or S; Q56 is G, R,or L; D110 is S, N, or G; and N111 is G or R. In some such cases thevariant further includes mutations at positions where P57 is A, K, G orL; and M60 is L or R. In some such cases the variant further includes amutation at position S105 where S105 is A, N, or D.

In some cases a subject DR IL-18 variant (i) includes mutationsC38S/C68S, C38S/C68G, C38S/C68A, C38S/C68D, or C38S/C68N (relative toSEQ ID NO: 30); (ii) has 90% or more sequence identity (e.g., 92% ormore, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more,98% or more, or 99% or more sequence identity) with the wild type humanIL-18 amino acid sequence set forth as SEQ ID NO: 30; and (iii) includesmutations as positions M51, K53, Q56, D110, and N111 relative to SEQ IDNO: 30. In some such cases the variant further includes mutations atpositions P57 and M60. In some such cases the variant further includes amutation at position S105.

In some cases a subject DR IL-18 variant (i) includes mutationsC38S/C68S, C38S/C68G, C38S/C68A, C38S/C68D, or C38S/C68N (relative toSEQ ID NO: 30); (ii) has 90% or more sequence identity (e.g., 92% ormore, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more,98% or more, or 99% or more sequence identity) with the wild type humanIL-18 amino acid sequence set forth as SEQ ID NO: 30; and (iii) includesmutations as positions M51, K53, Q56, D110, and N111 relative to SEQ IDNO: 30, where M51 is K; K53 is G or S; Q56 is G, R, or L; D110 is S, N,or G; and N111 is G or R. In some such cases the variant furtherincludes mutations at positions where P57 is A, K, G or L; and M60 is Lor R. In some such cases the variant further includes a mutation atposition S105 where S105 is A, N, or D.

In some cases a subject DR IL-18 variant (i) includes mutationsC38S/C68G, C38S/C68A, C38S/C68D, or C38S/C68N (relative to SEQ ID NO:30); (ii) has 90% or more sequence identity (e.g., 92% or more, 93% ormore, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more,or 99% or more sequence identity) with the wild type human IL-18 aminoacid sequence set forth as SEQ ID NO: 30; and (iii) includes mutationsas positions M51, K53, Q56, D110, and N111 relative to SEQ ID NO: 30. Insome such cases the variant further includes mutations at positions P57and M60. In some such cases the variant further includes a mutation atposition S105.

In some cases a subject DR IL-18 variant (i) includes mutationsC38S/C68G, C38S/C68A, C38S/C68D, or C38S/C68N (relative to SEQ ID NO:30); (ii) has 90% or more sequence identity (e.g., 92% or more, 93% ormore, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more,or 99% or more sequence identity) with the wild type human IL-18 aminoacid sequence set forth as SEQ ID NO: 30; and (iii) includes mutationsas positions M51, K53, Q56, D110, and N111 relative to SEQ ID NO: 30,where M51 is K; K53 is G or S; Q56 is G, R, or L; D110 is S, N, or G;and N111 is G or R. In some such cases the variant further includesmutations at positions where P57 is A, K, G or L; and M60 is L or R. Insome such cases the variant further includes a mutation at position S105where S105 is A, N, or D.

In some cases a subject DR IL-18 variant (i) includes mutation C38S/C68D(relative to SEQ ID NO: 30); (ii) has 90% or more sequence identity(e.g., 92% or more, 93% or more, 94% or more, 95% or more, 96% or more,97% or more, 98% or more, or 99% or more sequence identity) with thewild type human IL-18 amino acid sequence set forth as SEQ ID NO: 30;and (iii) includes mutations as positions M51, K53, Q56, D110, and N111relative to SEQ ID NO: 30. In some such cases the variant furtherincludes mutations at positions P57 and M60. In some such cases thevariant further includes a mutation at position S105.

In some cases a subject DR IL-18 variant (i) includes mutation C38S/C68D(relative to SEQ ID NO: 30); (ii) has 90% or more sequence identity(e.g., 92% or more, 93% or more, 94% or more, 95% or more, 96% or more,97% or more, 98% or more, or 99% or more sequence identity) with thewild type human IL-18 amino acid sequence set forth as SEQ ID NO: 30;and (iii) includes mutations as positions M51, K53, Q56, D110, and N111relative to SEQ ID NO: 30, where M51 is K; K53 is G or S; Q56 is G, R,or L; D110 is S, N, or G; and N111 is G or R. In some such cases thevariant further includes mutations at positions where P57 is A, K, G orL; and M60 is L or R. In some such cases the variant further includes amutation at position S105 where S105 is A, N, or D.

In some cases a subject DR IL-18 variant (i) includes mutation C38S/C68G(relative to SEQ ID NO: 30); (ii) has 90% or more sequence identity(e.g., 92% or more, 93% or more, 94% or more, 95% or more, 96% or more,97% or more, 98% or more, or 99% or more sequence identity) with thewild type human IL-18 amino acid sequence set forth as SEQ ID NO: 30;and (iii) includes mutations as positions M51, K53, Q56, D110, and N111relative to SEQ ID NO: 30. In some such cases the variant furtherincludes mutations at positions P57 and M60. In some such cases thevariant further includes a mutation at position S105.

In some cases a subject DR IL-18 variant (i) includes mutation C38S/C68G(relative to SEQ ID NO: 30); (ii) has 90% or more sequence identity(e.g., 92% or more, 93% or more, 94% or more, 95% or more, 96% or more,97% or more, 98% or more, or 99% or more sequence identity) with thewild type human IL-18 amino acid sequence set forth as SEQ ID NO: 30;and (iii) includes mutations as positions M51, K53, Q56, D110, and N111relative to SEQ ID NO: 30, where M51 is K; K53 is G or S; Q56 is G, R,or L; D110 is S, N, or G; and N111 is G or R. In some such cases thevariant further includes mutations at positions where P57 is A, K, G orL; and M60 is L or R. In some such cases the variant further includes amutation at position S105 where S105 is A, N, or D.

In some cases a subject DR IL-18 variant (i) includes mutation C38S/C68A(relative to SEQ ID NO: 30); (ii) has 90% or more sequence identity(e.g., 92% or more, 93% or more, 94% or more, 95% or more, 96% or more,97% or more, 98% or more, or 99% or more sequence identity) with thewild type human IL-18 amino acid sequence set forth as SEQ ID NO: 30;and (iii) includes mutations as positions M51, K53, Q56, D110, and N111relative to SEQ ID NO: 30. In some such cases the variant furtherincludes mutations at positions P57 and M60. In some such cases thevariant further includes a mutation at position S105.

In some cases a subject DR IL-18 variant (i) includes mutation C38S/C68A(relative to SEQ ID NO: 30); (ii) has 90% or more sequence identity(e.g., 92% or more, 93% or more, 94% or more, 95% or more, 96% or more,97% or more, 98% or more, or 99% or more sequence identity) with thewild type human IL-18 amino acid sequence set forth as SEQ ID NO: 30;and (iii) includes mutations as positions M51, K53, Q56, D110, and N111relative to SEQ ID NO: 30, where M51 is K; K53 is G or S; Q56 is G, R,or L; D110 is S, N, or G; and N111 is G or R. In some such cases thevariant further includes mutations at positions where P57 is A, K, G orL; and M60 is L or R. In some such cases the variant further includes amutation at position S105 where S105 is A, N, or D.

In some cases a subject DR IL-18 variant (i) includes mutation C38S/C68N(relative to SEQ ID NO: 30); (ii) has 90% or more sequence identity(e.g., 92% or more, 93% or more, 94% or more, 95% or more, 96% or more,97% or more, 98% or more, or 99% or more sequence identity) with thewild type human IL-18 amino acid sequence set forth as SEQ ID NO: 30;and (iii) includes mutations as positions M51, K53, Q56, D110, and N111relative to SEQ ID NO: 30. In some such cases the variant furtherincludes mutations at positions P57 and M60. In some such cases thevariant further includes a mutation at position S105.

In some cases a subject DR IL-18 variant (i) includes mutation C38S/C68N(relative to SEQ ID NO: 30); (ii) has 90% or more sequence identity(e.g., 92% or more, 93% or more, 94% or more, 95% or more, 96% or more,97% or more, 98% or more, or 99% or more sequence identity) with thewild type human IL-18 amino acid sequence set forth as SEQ ID NO: 30;and (iii) includes mutations as positions M51, K53, Q56, D110, and N111relative to SEQ ID NO: 30, where M51 is K; K53 is G or S; Q56 is G, R,or L; D110 is S, N, or G; and N111 is G or R. In some such cases thevariant further includes mutations at positions where P57 is A, K, G orL; and M60 is L or R. In some such cases the variant further includes amutation at position S105 where S105 is A, N, or D.

In some embodiments, an IL-18 polypeptide (e.g., DR IL-18) disclosedherein does not contain a cysteine at residue 78 (e.g., relative to SEQID NO: 30, 89, and/or 19). In some embodiments, an IL-18 polypeptide(e.g., DR IL-18) disclosed herein does not contain a mutation at residueN78 (e.g., relative to SEQ ID NO: 30, 89, and/or 19).

In some embodiments, an IL-18 polypeptide (e.g., DR IL-18) disclosedherein does not contain a cysteine at residue 121 (e.g., relative to SEQID NO: 30, 89, and/or 19). In some embodiments, an IL-18 polypeptide(e.g., DR IL-18) disclosed herein does not contain a mutation at residueE121 (e.g., relative to SEQ ID NO: 30, 89, and/or 19).

In some embodiments, an IL-18 polypeptide (e.g., DR IL-18) disclosedherein does not contain a cysteine at residue 144 (e.g., relative to SEQID NO: 30, 89, and/or 19). In some embodiments, an IL-18 polypeptide(e.g., DR IL-18) disclosed herein does not contain a mutation at residueL144 (e.g., relative to SEQ ID NO: 30, 89, and/or 19).

In some embodiments, an IL-18 polypeptide (e.g., DR IL-18) disclosedherein does not contain a cysteine at residue 157 (e.g., relative to SEQID NO: 30, 89, and/or 19). In some embodiments, an IL-18 polypeptide(e.g., DR IL-18) disclosed herein does not contain a mutation at residueD157 (e.g., relative to SEQ ID NO: 30, 89, and/or 19).

In some embodiments, an IL-18 polypeptide (e.g., DR IL-18) disclosedherein does not contain a cysteine at residue 78, residue 121, residue144, or residue 157 (e.g., relative to SEQ ID NO: 30, 89, and/or 19). Insome embodiments, an IL-18 polypeptide (e.g., DR IL-18) disclosed hereindoes not contain a mutation at residue N78, residue E121, residue L144,or residue D157 (e.g., relative to SEQ ID NO: 30, 89, and/or 19).

An IL-18 polypeptide (e.g., DR IL-18) of the disclosure can comprisemutations to C38 and C68 as disclosed herein, and can lack any mutationsthat introduce non-native cysteine residues into the polypeptidesequence. For example, in some embodiments, an IL-18 polypeptide (e.g.,DR IL-18) contains cysteines only at position 76 and/or 127 (e.g.,relative to SEQ ID NO: 30, 89, or 19). In some embodiments, an IL-18polypeptide (e.g., DR IL-18) does not contain any cysteines except atposition(s) 76 and/or 127 (e.g., relative to SEQ ID NO: 30, 89, or 19).A lack of non-native cysteine residues can contribute to advantageousproperties of IL-18 polypeptides of the disclosure, for example, becausecysteine residues can negatively impact the stability and/or biologicalactivity of IL-18, e.g., by allowing formation of intramolecular orintermolecular disulfide bonds, dimers, aggregates, and the like.

An IL-18 polypeptide (e.g., DR IL-18) of the disclosure can lack orsubstantially-lack surface-exposed or solvent-exposed cysteine residues.A lack of surface-exposed or solvent-exposed cysteine residues cancontribute to advantageous properties of IL-18 polypeptides of thedisclosure, for example, because surface-exposed or solvent-exposedcysteine residues can negatively impact the stability and/or biologicalactivity of IL-18, e.g., by allowing formation of intramolecular orintermolecular disulfide bonds, dimers, aggregates, and the like. AnIL-18 polypeptide (e.g., DR IL-18) of the disclosure can lack orsubstantially-lack surface-exposed or solvent-exposed cysteine residuesas determined by, for example, structural modeling, or an assay todetect free thiols (such as Ellman's titration). An IL-18 polypeptide(e.g., DR IL-18) of the disclosure can lack or substantially-lack freeor surface-exposed thiols.

An IL-18 polypeptide disclosed herein (e.g., a DR IL-18) can becharacterized by its molecular weight. For example, an IL-18 polypeptidecan have a molecular weight of about 18 kDa. In some embodiments, anIL-18 polypeptide disclosed herein has a molecular weight of about 13kDa, about 14 kDa, about 15 kDa, about 16 kDa, about 17 kDa, about 18kDa, about 19 kDa, about 20 kDa, about 21 kDa, about 22 kDa, or about 23kDa. In some embodiments, an IL-18 polypeptide disclosed herein has amolecular weight of at least about 10 kDa, at least about 11 kDa, atleast about 12 kDa, at least about 13 kDa, at least about 14 kDa, atleast about 15 kDa, at least about 16 kDa, at least about 17 kDa, atleast about 18 kDa, at least about 19 kDa, or at least about 20 kDa. Insome embodiments, an IL-18 polypeptide disclosed herein has a molecularweight of at most about 17 kDa, at most about 18 kDa, at most about 19kDa, at most about 20 kDa, at most about 21 kDa, at most about 22 kDa,at most about 23 kDa, at most about 24 kDa, at most about 25 kDa, atmost about 30 kDa, at most about 40 kDa, at most about 50 kDa, or atmost about 100 kDa. In some embodiments, an IL-18 polypeptide disclosedherein has a molecular weight of about 18.15153 kDa, about 18.1515 kDa,about 18.152 kDa, about 18.15 kDa, about 18.2 kDa, or about 18 kDa.

An IL-18 polypeptide disclosed herein (e.g., a DR IL-18) can comprise,for example, about 130, about 135, about 140, about 145, about 150,about 151, about 152, about 153, about 154, about 155, about 156, about157, about 158, about 159, about 160, about 161, about 162, about 163,about 1645, about 165, about 170, about 175, about 180, about 185, orabout 190 amino acids.

In some embodiments, an IL-18 polypeptide disclosed herein (e.g., a DRIL-18) comprises at least about 130, at least about 135, at least about140, at least about 145, at least about 150, at least about 151, atleast about 152, at least about 153, at least about 154, at least about155, at least about 156, at least about 157, at least about 158, atleast about 159, or at least about 160 amino acids. In some embodiments,an IL-18 polypeptide disclosed herein (e.g., a DR IL-18) comprises atmost about 155, at most about 156, at most about 157, at most about 158,at most about 159, at most about 160, at most about 161, at most about162, at most about 163, at most about 1645, at most about 165, at mostabout 170, at most about 175, at most about 180, at most about 185, orat most about 190 amino acids. In some embodiments, an IL-18 polypeptidedisclosed herein (e.g., a DR IL-18) comprises about 150 to about 170,about 152 to about 162, about 155 to about 159, about 156 to about 158,or 157 amino acids.

An IL-18 polypeptide disclosed herein (e.g., a DR IL-18, such as a DRIL-18 in a formulation of the disclosure) can have a level of biologicalactivity or potency that can be measured by an assay for biologicalactivity. A non-limiting example of an assay that can be used to measurea level of potency or biological activity of an IL-18 polypeptidedisclosed herein is an IL-18 HEK-Blue potency assay. HEK-Blue IL-18cells are designed to detect bioactive IL-18 by monitoring activation ofthe NFκβ and AP-1 pathways. These reporter cells were generated bystable transfection of HEK293-derived cells with genes encoding IL-18Rαand IL-18Rβ. Responses to human TNF-α and IL-1β have also been blocked,so the cells specifically respond to IL-18. These cells express asecreted embryonic alkaline phosphatase (SEAP) reporter gene undercontrol of the IFN-β minimal promoter fused to five NFκβ and five AP-1binding sites. Binding of bioactive IL-18 to the heterodimeric IL-18receptor on the surface of HEK-Blue IL-18 cells triggers a signalingcascade that leads to production of SEAP, which can be quantified with acolorimetric assay.

The biological activity or potency of an IL-18 polypeptide disclosedherein (e.g., a DR IL-18, such as a DR IL-18 in a formulation of thedisclosure) can be compared to a reference, for example, a wild typeIL-18, or a reference standard of the same IL-18 polypeptide producedand characterized previously. In some embodiments, an IL-18 polypeptidedisclosed herein (e.g., a DR IL-18, such as a DR IL-18 in a formulationof the disclosure) has a biological activity or potency (e.g., EC50)that is at least 5%, at least 10%, at least 15%, at least 20%, at least25%, at least 30%, at least 35%, at least 40%, at least 45%, at least50%, at least 55%, at least 60%, at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, or at least 95% of thereference value. In some embodiments, an IL-18 polypeptide disclosedherein (e.g., a DR IL-18, such as a DR IL-18 in a formulation of thedisclosure) has a biological activity or potency (e.g., EC50) that is atmost 105%, at most 110%, at most 115%, at most 120%, at most 125%, atmost 130%, at most 135%, at most 140%, at most 145%, at most 150%, atmost 155%, at most 160%, at most 165%, at most 170%, at most 175%, atmost 180%, at most 185%, at most 190%, at most 195%, at most 200%, atmost 250%, or at most 300% of the reference value. In some embodiments,an IL-18 polypeptide disclosed herein (e.g., a DR IL-18, such as a DRIL-18 in a formulation of the disclosure) has a biological activity orpotency (e.g., EC50) that is about 30% to about 500%, about 30% to about250%, about 30% to about 200%, about 30% to about 175%, about 30% toabout 150%, about 30% to about 140%, about 30% to about 130%, about 30%to about 120%, about 30% to about 110%, about 30% to about 105%, about50% to about 500%, about 50% to about 250%, about 50% to about 200%,about 50% to about 175%, about 50% to about 150%, about 50% to about140%, about 50% to about 130%, about 50% to about 120%, about 50% toabout 110%, about 50% to about 105%, about 60% to about 500%, about 60%to about 260%, about 60% to about 200%, about 60% to about 175%, about60% to about 160%, about 60% to about 140%, about 60% to about 130%,about 60% to about 120%, about 60% to about 110%, about 60% to about105%, about 70% to about 500%, about 70% to about 250%, about 70% toabout 200%, about 70% to about 175%, about 70% to about 150%, about 70%to about 140%, about 70% to about 130%, about 70% to about 120%, about70% to about 110%, about 70% to about 105%, about 90% to about 500%,about 90% to about 250%, about 90% to about 200%, about 90% to about175%, about 90% to about 150%, about 90% to about 140%, about 90% toabout 130%, about 90% to about 120%, about 90% to about 110%, or about90% to about 105% of the reference value. In some embodiments, thebiological activity or potency (e.g., EC50) at least 60% and at most140% of the reference value.

In some embodiments, an IL-18 polypeptide disclosed herein (e.g., a DRIL-18, such as a DR IL-18 in a formulation of the disclosure) has abiological activity or potency (e.g., EC50) that is at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, or atleast 95%, at least 97.5%, at least 100%, at least 102.5%, at least105%, at least 110%, at least 115%, at least 120%, at least 125%, atleast 130%, at least 140%, at least 150%, or at least 200%, at least3-fold, at least 4-fold, at least 5-fold, at least 6-fold, atleast-fold, at least 8-fold, at least 9-fold, or at least 10-fold morepotent than of the biological activity of human wild type IL-18, e.g.,as determined by an IL-18 HEK-Blue potency assay.

In some embodiments, an IL-18 polypeptide disclosed herein exhibits anEC50 of less than about 100 μM, less than about 10 μM, less than about 1μM, less than about 900 nM, less than about 800 nM, less than about 700nM, less than about 600 nM, less than about 500 nM, less than about 400nM, less than about 300 nM, less than about 200 nM, less than about 100nM, less than about 50 nM, less than about 30 nM, less than about 10 nM,less than about 5 nM, less than about 4 nM, less than about 3 nM, lessthan about 2 nM, less than about 1 nM, less than about 900 pM, less thanabout 800 pM, less than about 700 pM, less than about 600 pM, less thanabout 500 pM, less than about 400 pM, less than about 300 pM, less thanabout 200 pM, less than about 100 pM, less than about 50 pM, less thanabout 10 pM, or less than about 1 pM, e.g., as determined by an IL-18HEK-Blue potency assay.

In some embodiments, an IL-18 polypeptide disclosed herein exhibits anEC50 of about 1 pM to about 1 μM, about 1 pM to about 500 nM, about 1 pMto about 400 nM, about 1 pM to about 300 nM, about 1 pM to about 200 nM,about 1 pM to about 100 nM, about 1 pM to about 50 nM, about 1 pM toabout 10 pM, about 10 pM to about 1 μM, about 10 pM to about 500 nM,about 10 pM to about 400 nM, about 10 pM to about 300 nM, about 10 pM toabout 200 nM, about 10 pM to about 100 nM, about 10 pM to about 50 nM,about 50 pM to about 1 μM, about 50 pM to about 500 nM, about 50 pM toabout 400 nM, about 50 pM to about 300 nM, about 50 pM to about 200 nM,about 50 pM to about 100 nM, about 100 pM to about 1 μM, about 100 pM toabout 500 nM, about 100 pM to about 400 nM, about 100 pM to about 300nM, or about 100 pM to about 200 nM, for example, as determined by anIL-18 HEK-Blue potency assay.

Nucleic Acids

Also provided herein are nucleic acids encoding any of the proteinsdescribed herein (e.g., IL-18 protein having a mutation at C38 and C68).Such nucleic acids can take any convenient form (e.g. a PCR product, avector such as a viral vector, plasmid, cosmid, minicircle, and thelike). In some cases the nucleotide sequence encoding the subject IL-18protein is operably linked to (under the control of) a promoter (e.g.,that is functional in a prokaryotic cell, or in some cases that isfunctional in a eukaryotic cell such as yeast or immune cells such as Tcells, NK cells, TILs, and the like).

Also provided herein are cells comprising such nucleic acids and/orcomprising the subject IL-18 proteins (e.g., stabilized proteins). Insome cases the cell is a prokaryotic cell such as a bacterial cell(e.g., for the purpose of propagating the nucleic acid or for thepurpose of producing a subject protein), in some cases the cell is aeukaryotic such as a yeast cell (e.g., for the purpose of producing asubject protein), and in some cases the cell is an immune cell such as aT-cell (e.g., a CAR-T cell such as a TRUCK cell), TIL, TCR-T cell (a Tcell with an engineered TCR, e.g., engineered to bind to a targetantigen with increased affinity compared to a natural TCR), or NK-cell(e.g., a CAR-NK cell), e.g., such cells can be engineered to express(e.g., secrete) a subject IL-18 protein (e.g., stabilized proteins suchas SEQ ID NO: 19).

Cytokine Inhibitors

In some embodiments, the composition of the present disclosure comprisesan inhibitor of one or more cytokines. In some embodiments, theinhibitor of one or more cytokines comprises a chemical compound, aprotein, a peptide, a peptidomimetic, an antibody, a ribozyme, a smallmolecule chemical compound, or an antisense nucleic acid molecule (e.g.,siRNA, miRNA, etc.) that inhibits the expression, activity, or both ofone or more cytokines. In some embodiments, the inhibitor inhibits theexpression, activity, or both of IL-17, IL-5, or IL-3. In someembodiments, the cytokine inhibitor decreases toxicity. In someembodiments, the cytokine inhibitor increases efficacy of anadministered IL-18 variant polypeptide (e.g., stabilized IL-18 variants,DR IL-18 variants, D2D IL-18 variants, stabilized DR IL-18 variants, andstabilized D2D IL-18 variants).

Treatment

In various embodiments, the present disclosure includes compositionscomprising an activator of IL-18 activity (e.g., stabilized IL-18variants, DR IL-18 variants, D2D IL-18 variants, stabilized DR IL-18variants, and stabilized D2D IL-18 variants) for use in methods such asstimulating signaling activity through at least one IL-18R, in a cell,tissue, organ, system, or subject in need thereof. In variousembodiments, the activator of IL-18 activity compositions, and methodsof treatment of the invention, increase the amount of IL-18R signaling,the amount of immune cell activity, or both. In various embodiments, thediseases and disorders in which an increase in IL-18R signaling mayimprove therapeutic outcomes include, but are not limited to cancer,infectious diseases, macular degeneration, and metabolic diseases ordisorders.

Various IL-18 polypeptides are discussed above (e.g., IL-18 polypeptideswith C38/C68 mutations) and are not repeated here—because any of theproteins discuss above can be used in the methods discussed herein. Forexample, in some cases a stabilized IL-18 polypeptide (e.g., stabilizedIL-18 variant polypeptides such as stabilized DR IL-18 variants andstabilized D2D IL-18 variants) is used, e.g., to treat a disease ordisorder such as cancer or an infection (e.g., a cancer that isresistant to immune checkpoint inhibitors, a cancer associated with atumor that has reduced or absent expression of MHC class I, a cancerassociated with high levels of IL-18BP (e.g., circulating or expressedby tumors), a cancer associated with a tumor in which IL-18R isexpressed on infiltrating T cells or NK cells, a metabolic disease ordisorder, an infectious disease, a viral infection such as HIV, and thelike).

In some cases, the administered polypeptide is a wild type IL-18polypeptide (e.g., a wild type human IL-18 protein). In some cases, theadministered polypeptide is a subject stabilized IL-18 polypeptide(e.g., one that includes amino acid mutations at positions C38 and C68relative to SEQ ID NO: 30). In some cases, the administered polypeptideis an IL-18 variant polypeptide such as a DR IL-18 variant or a D2DIL-18 variant. In some cases, the administered polypeptide is astabilized IL-18 variant polypeptide (e.g., a stabilized DR IL-18variant or a stabilized D2D IL-18 variant—see, e.g., the stabilized DRIL-18 variant polypeptides discussed above).

Fusions/Conjugations

In some embodiments, an IL-18 polypeptide (e.g., a stabilized IL-18 suchas a stabilized DR IL-18 variant or stabilized D2D IL-18 variant, andthe like) (e.g., SEQ ID NO: 19) of the present disclosure is conjugatedto another molecule (e.g., fused to another protein), i.e., an IL-18variant polypeptide can be fused in frame with a second polypeptide (afusion partner). In some embodiments, the second polypeptide (the fusionpartner) is capable of increasing the overall size of the fusionprotein, e.g., so that the fusion protein will not be cleared from thecirculation rapidly. In some cases, an IL-18 polypeptide (e.g., astabilized IL-18 such as a stabilized DR IL-18 variant or stabilized D2DIL-18 variant, and the like) (e.g., SEQ ID NO: 19) is not fused to asecond polypeptide.

In some embodiments, the second polypeptide (the fusion partner for aIL-18 variant polypeptide or a fragment thereof) is part or whole of animmunoglobulin Fc region (i.e., an antibody Fc sequence). In otherembodiments, the second polypeptide is any suitable polypeptide that issubstantially similar to Fc, e.g., providing increased size,multimerization domains, and/or additional binding or interaction withIg molecules. In some embodiments, the second polypeptide is part orwhole of Human Serum Albumin (HSA). In some embodiments, the secondpolypeptide is part or whole of an antibody, antibody fragment, camelidantibody or “nanobody” or other affinity reagent that binds to orinteracts with HSA. These fusion proteins can facilitate purification,multimerization, and show an increased half-life in vivo. Fusionproteins having disulfide-linked multimeric structures can also, in somecases, be more efficient in binding and neutralizing other molecules.

When fused to a heterologous polypeptide, the portion corresponding tothe IL-18 polypeptide (e.g., a stabilized IL-18 such as a stabilized DRIL-18 variant or stabilized D2D IL-18 variant, and the like) (e.g., SEQID NO: 19) can be referred to as the “IL-18 variant polypeptide portion”of a subject IL-18 variant polypeptide.

In some cases, the second polypeptide is a marker sequence (e.g., anaffinity tag), such as a peptide that facilitates purification of thefused polypeptide. For example, the marker amino acid sequence can be ahexa-histidine peptide, such as the tag provided in a pQE vector(QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), amongothers, many of which are commercially available. As described in Gentzet al., Proc. Natl. Acad. Sci. USA 86: 821-824, 1989, for instance,hexa-histidine provides for convenient purification of the fusionprotein. Another peptide tag useful for purification, the “HA” tag,corresponds to an epitope derived from the influenza hemagglutininprotein. Wilson et al., Cell 37: 767, 1984. The addition of peptidemoieties to facilitate handling of polypeptides are familiar and routinetechniques in the art. A subject IL-18 polypeptide (e.g., a stabilizedIL-18 such as a stabilized DR IL-18 variant or stabilized D2D IL-18variant, and the like) (e.g., SEQ ID NO: 19) can be modified, e.g.,joined/conjugated to a wide variety of other oligopeptides, proteins,and/or non-protein moieties for a variety of purposes. For example,post-translationally modified, for example by prenylation, acetylation,amidation, carboxylation, glycosylation, PEGylation (covalent attachmentof polyethylene glycol (PEG) polymer chains), etc. Such modificationscan also include modifications of glycosylation, e.g. those made bymodifying the glycosylation patterns of a polypeptide during itssynthesis and processing or in further processing steps; e.g. byexposing the polypeptide to enzymes which affect glycosylation, such asmammalian glycosylating or deglycosylating enzymes. In some embodiments,a subject IL-18 variant polypeptide has one or more phosphorylated aminoacid residues, e.g. phosphotyrosine, phosphoserine, or phosphothreonine.

In some embodiments, an IL-18 polypeptide disclosed herein isglycosylated. In some embodiments, an IL-18 polypeptide disclosed hereinis non-glycosylated.

In some embodiments, an IL-18 polypeptide disclosed herein is conjugatedto a water-soluble polymer. In some embodiments, an IL-18 polypeptidedisclosed herein is pegylated. In some embodiments, an IL-18 polypeptidedisclosed herein is conjugated to a polyethylene glycol homopolymer,polyethylene glycol copolymer, polypropylene glycol homopolymer,poly(N-vinylpyrrolidone), poly(vinyl alcohol), poly(ethyleneglycol-co-propylene glycol), poly(N-2-(hydroxypropyl)methacrylamide),poly(sialic acid), poly(N-acryloyl morpholine), or dextran.

In some embodiments, an IL-18 polypeptide disclosed herein is notconjugated to a water-soluble polymer. In some embodiments, an IL-18polypeptide disclosed herein is not pegylated. In some embodiments, anIL-18 polypeptide disclosed herein is not conjugated to a polyethyleneglycol homopolymer, polyethylene glycol copolymer, polypropylene glycolhomopolymer, poly(N-vinylpyrrolidone), poly(vinyl alcohol),poly(ethylene glycol-co-propylene glycol),poly(N-2-(hydroxypropyl)methacrylamide), poly(sialic acid),poly(N-acryloyl morpholine), or dextran.

In some other embodiments, an IL-18 polypeptide (e.g., a stabilizedIL-18 such as a stabilized DR IL-18 variant or stabilized D2D IL-18variant, and the like) (e.g., SEQ ID NO: 19) of the disclosure includereagents further modified to improve their resistance to proteolyticdegradation or to optimize solubility properties or to render them moresuitable as a therapeutic agent. For example, variants of the presentdisclosure further include analogs containing residues other thannaturally occurring L-amino acids, e.g. D-amino acids or non-naturallyoccurring synthetic amino acids. D-amino acids may be substituted forsome or all of the amino acid residues.

Co-Administration and Multispecific IL-18 Variant Polypeptides

In some cases a subject IL-18 polypeptide (e.g., a stabilized IL-18 suchas a stabilized DR IL-18 variant or stabilized D2D IL-18 variant, andthe like) (e.g., SEQ ID NO:19) is administered with an additional agent.The terms “co-administration”, “co-administer”, and “in combinationwith” include the administration of two or more therapeutic agents(e.g., a subject stabilized IL-18) either simultaneously, concurrentlyor sequentially within no specific time limits. In some embodiments, theagents are present in the cell or in the subject's body at the same timeor exert their biological or therapeutic effect at the same time. Insome embodiments, the therapeutic agents are in the same composition orunit dosage form. In other embodiments, the therapeutic agents are inseparate compositions or unit dosage forms. In certain embodiments, afirst agent can be administered prior to (e.g., minutes, 15 minutes, 30minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks,5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, orsubsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours,96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks,or 12 weeks after) the administration of a second therapeutic agent.

In some cases, a subject IL IL-18 polypeptide (e.g., a stabilized IL-18such as a stabilized DR IL-18 variant or stabilized D2D IL-18 variant,and the like) (e.g., SEQ ID NO: 19) (e.g., formulated as apharmaceutical composition) is co-administered with a cancer therapeuticdrug, therapeutic drug to treat an infection, or cancer-directedantibody. Such administration may involve concurrent (i.e. at the sametime), prior, or subsequent administration of the drug/antibody withrespect to the administration of an agent or agents of the disclosure. Aperson of ordinary skill in the art would have no difficulty determiningthe appropriate timing, sequence and dosages of administration forparticular drugs and compositions of the present disclosure.

In some embodiments, treatment is accomplished by administering acombination (co-administration) of a subject IL-18 polypeptide (e.g., astabilized IL-18 such as a stabilized DR IL-18 variant or stabilized D2DIL-18 variant, and the like) (e.g., SEQ ID NO: 19) with another agent(e.g., an immune stimulant, an agent to treat chronic infection, acytotoxic agent, an anti-cancer agent, etc.). One example class ofcytotoxic agents that can be used are chemotherapeutic agents. Exemplarychemotherapeutic agents include, but are not limited to, aldesleukin,altretamine, amifostine, asparaginase, bleomycin, capecitabine,carboplatin, carmustine, cladribine, cisapride, cisplatin,cyclophosphamide, cytarabine, dacarbazine (DTIC), dactinomycin,docetaxel, doxorubicin, dronabinol, duocarmycin, etoposide, filgrastim,fludarabine, fluorouracil, gemcitabine, granisetron, hydroxyurea,idarubicin, ifosfamide, interferon alpha, irinotecan, lansoprazole,levamisole, leucovorin, megestrol, mesna, methotrexate, metoclopramide,mitomycin, mitotane, mitoxantrone, omeprazole, ondansetron, paclitaxel(Taxol™), pilocarpine, prochloroperazine, rituximab, saproin, tamoxifen,taxol, topotecan hydrochloride, trastuzumab, vinblastine, vincristineand vinorelbine tartrate.

A subject IL-18 polypeptide (e.g., a stabilized IL-18 such as astabilized DR IL-18 variant or stabilized D2D IL-18 variant, and thelike) (e.g., SEQ ID NO: 19) need not be, but is optionally formulatedwith one or more agents that potentiate activity, or that otherwiseincrease the therapeutic effect. In some embodiments, treatment isaccomplished by administering a combination (co-administration) of asubject IL-18 polypeptide (e.g., a stabilized IL-18 such as a stabilizedDR IL-18 variant or stabilized D2D IL-18 variant, and the like) (e.g.,SEQ ID NO: 19) and an agent that opsonizes a target cell. Thus, alsoenvisioned herein are compositions (and methods that use thecompositions) that include: (a) a subject IL-18 polypeptide (e.g., astabilized IL-18 such as a stabilized DR IL-18 variant or stabilized D2DIL-18 variant, and the like) (e.g., SEQ ID NO: 19); and (b) an agentthat opsonizes the target cell. In some cases, that agent that opsonizesthe target cell is Rituximab. In some cases, that agent that opsonizesthe target cell is Cetuximab.

An “agent that opsonizes a target cell” (an “opsonizing agent”) is anyagent that can bind to a target cell (e.g., a cancer cell, a cellharboring an intracellular pathogen, etc.) and opsonize the target cell(e.g., mark the target cell for phagocytosis and/or forantibody-dependent cell mediated cytotoxicity (ADCC)). For example, anyantibody that can bind to a target cell (e.g., a cancer cell such as atumor cell), where the antibody has an FC region, is considered to be anagent that opsonizes a target cell. In some cases, the agent thatopsonizes a target cell is an antibody that binds to a target cell(e.g., an anti-tumor antibody, an anti-cancer antibody, ananti-infection antibody, and the like).

For example antibodies selective for tumor cell markers, radiation,surgery, and/or hormone deprivation, see Kwon et al., Proc. Natl. Acad.Sci U.S.A., 96: 15074-9, 1999.

Angiogenesis inhibitors can also be combined with the methods of theinvention. A number of antibodies are currently in clinical use for thetreatment of cancer, and others are in varying stages of clinicaldevelopment. For example, there are a number of antigens andcorresponding monoclonal antibodies for the treatment of B cellmalignancies. One target antigen is CD20. Rituximab is a chimericunconjugated monoclonal antibody directed at the CD20 antigen. CD20 hasan important functional role in B cell activation, proliferation, anddifferentiation. The CD52 antigen is targeted by the monoclonal antibodyalemtuzumab, which is indicated for treatment of chronic lymphocyticleukemia. CD22 is targeted by a number of antibodies, and has recentlydemonstrated efficacy combined with toxin in chemotherapy-resistanthairy cell leukemia. Two new monoclonal antibodies targeting CD20,tositumomab and ibritumomab, have been submitted to the Food and DrugAdministration (FDA). These antibodies are conjugated withradioisotopes. Alemtuzumab (Campath) is used in the treatment of chroniclymphocytic leukemia; Gemtuzumab (Mylotarg) finds use in the treatmentof acute myelogenous leukemia; Ibritumomab (Zevalin) finds use in thetreatment of non-Hodgkin's lymphoma; Panitumumab (Vectibix) finds use inthe treatment of colon cancer.

Monoclonal antibodies useful in the methods of the disclosure that havebeen used in solid tumors include, without limitation, edrecolomab andtrastuzumab (herceptin). Edrecolomabtargets the 17-1A antigen seen incolon and rectal cancer, and has been approved for use in Europe forthese indications. Trastuzumab targets the HER-2/neu antigen. Cetuximab(Erbitux) is also of interest for use in the methods of the disclosure.The antibody binds to the EGF receptor (EGFR), and has been used in thetreatment of solid tumors including colon cancer and squamous cellcarcinoma of the head and neck (SCCHN).

A subject IL-18 polypeptide (e.g., a stabilized IL-18 such as astabilized DR IL-18 variant or stabilized D2D IL-18 variant, and thelike) can be combined with any of the above mentioned agents (e.g.,agents such as antibodies that opsonize a target cell). Thus, in somecases, a subject IL-18 polypeptide (e.g., a stabilized IL-18 such as astabilized DR IL-18 variant or stabilized D2D IL-18 variant, and thelike) is used in a combination therapy (is co-administered) with one ormore opsonizing agents selective for cancer cells, e.g., tumor cells. Insome cases, a subject IL-18 polypeptide (e.g., a stabilized IL-18 suchas a stabilized DR IL-18 variant or stabilized D2D IL-18 variant, andthe like) is used in a combination therapy (is co-administered) with oneor more of: cetuximab (binds EGFR), panitumumab (binds EGFR), rituximab(binds CD20), trastuzumab (binds HER2), pertuzumab (binds HER2),alemtuzumab (binds CD52), brentuximab (binds CD30), tositumomab,ibritumomab, gemtuzumab, ibritumomab, and edrecolomab (binds 17-1A), ora combination thereof.

In some cases, a subject IL-18 polypeptide (e.g., a stabilized IL-18such as a stabilized DR IL-18 variant or stabilized D2D IL-18 variant,and the like) (e.g., SEQ ID NO: 19) is co-administered with a cancercell opsonizing agent (e.g., one that comprises an antigen bindingregion that targets CD19, CD20, CD22, CD24, CD25, CD30, CD33, CD37,CD38, CD44, CD45, CD47, CD51, CD52, CD56, CD62L, CD70, CD74, CD79, CD80,CD96, CD97, CD99, CD123, CD134, CD138, CD152 (CTLA-4), CD200, CD213A2,CD221, CD248, CD276 (B7H3), B7H4, CD279 (PD-1), CD274 (PD-L1), CD319,SIRPa, EGFR, EPCAM, 17-1A, HER1, HER2, HER3, CD117, C-Met, HGFR, PDGFRA,AXL, TWEAKR, PTHR2, HAVCR2 (TIM3), GD2 ganglioside, MUC1, mucin CanAg,mesothelin, endoglin, Lewis-Y antigen, CEA, CEACAM1, CEACAM5, CA-125,PSMA, BAFF, FGFR2, TAG-72, gelatinase B, glypican 3, nectin-4, BCMA,CSF1R, SLAMF7, integrin αvβ3, TYRP1, GPNMB, CLDN18.2, FOLR1, CCR4,CXCR4, MICA, C242 antigen, DLL3, DLL4, EGFL7, vimentin, fibronectinextra domain-B, TROP-2, LRRC15, FAP, SLITRK6, NOTCH2, NOTCH3,Tenascin-3, STEAP1, or NRP1, or any combination thereof).

In some cases, a subject IL-18 polypeptide (e.g., a stabilized IL-18such as a stabilized DR IL-18 variant or stabilized D2D IL-18 variant,and the like) (e.g., SEQ ID NO: 19) is co-administered with and agentthat targets one or more antigens selected from: CD19, CD20, CD22, CD24,CD25, CD30, CD33, CD38, CD44, CD47, SIRPA, CD52, CD56, CD70, CD96, CD97,CD99, CD123, CD279 (PD-1), CD274 (PD-L1), EGFR, 17-1A, HER2, CD117,C-Met, PTHR2, and HAVCR2 (TIM3).

In some cases, a subject IL-18 polypeptide (e.g., a stabilized IL-18such as a stabilized DR IL-18 variant or stabilized D2D IL-18 variant,and the like) (e.g., SEQ ID NO: 19) is used in a combination therapy (isco-administered) with a convenient immunomodulatory agent (e.g., animmune checkpoint inhibitor or immune agonist). As noted above, examplesof targets of immune checkpoint inhibitors include but are not limitedto: PD-L1, PD1, CTLA4, TIM3, TIGIT, LAG3, B7H3, B7H4, VISTA, BTLA, CD47,SIRP alpha, CD48, CD155, CD160, TREM2, IDO1, Adenosine 2A receptor, Arylhydrocarbon receptor, KIR, and LILRB2. Thus, examples of immunecheckpoint inhibitors include agents (e.g., antibodies) that inhibitproteins such as: PD-L1, PD1, CTLA4, TIM3, TIGIT, LAG3, B7H3, B7H4,VISTA, BTLA, CD47, SIRP alpha, CD48, CD155, CD160, TREM2, IDO1,Adenosine 2A receptor, Aryl hydrocarbon receptor, KIR, and LILRB2. Insome cases, a subject stabilized IL-18 variant polypeptide (e.g., aDR-IL-18 variant, a D2D-IL-18 variant) (e.g., SEQ ID NO: 19) isco-administered with an immune checkpoint inhibitor (e.g., an antibody)that inhibits PD-L1, PD1, CTLA4, TIM3, TIGIT, LAG3, B7H3, B7H4, VISTA,BTLA, CD47, SIRP alpha, CD48, CD155, CD160, TREM2, IDO1, Adenosine 2Areceptor, Aryl hydrocarbon receptor, KIR, LILRB2, or any combinationthereof.

In some cases, a subject stabilized IL-18 variant polypeptide (e.g., aDR-IL-18 variant, a D2D-IL-18 variant) (e.g., SEQ ID NO: 19) isco-administered with an immune agonist. Examples of immune agonistsinclude agents (e.g., agonizing antibodies) that agonize a protein suchas: a tumor necrosis factor receptor superfamily (TNFRSF) protein (e.g.,GITR, 41BB, OX40, CD27, CD40, HVEM); an immunoglobulin superfamily(IgSF) protein (e.g., CD28, ICOS, CD226, NKG2D); a TLR (e.g., TLR2,TLR4, TLR5, TLR7, TLR9); a nucleic acid sensor (e.g., STING, cGAS, RIG-I(DDX58)). Examples of immune agonists also include, but are not limitedto: inflammasome activators (agonists), T cell engagers (e.g., amultispecific agent that cross-links CD3 and/or CD28 with a tumorantigen, e.g., a BiTE), and cytokines or cytokine variants (e.g., IL-2,IL-7, IL-10, IL-12, IL-15, IL-21, IL-23, IL-27, IL-33, TNF, TL1A, IFNA,IFNB, IFNG).

Thus, In some cases, a subject stabilized IL-18 variant polypeptide(e.g., a DR-IL-18 variant, a D2D-IL-18 variant) (e.g., SEQ ID NO: 19) isco-administered with an immune agonist such as: an agent (e.g.,antibody) that agonizes a tumor necrosis factor receptor superfamily(TNFRSF) protein (e.g., GITR, 41BB, OX40, CD27, CD40, HVEM); an agent(e.g., antibody) that agonizes an immunoglobulin superfamily (IgSF)protein (e.g., CD28, ICOS, CD226, NKG2D); an agent that agonizes a TLR(e.g., TLR2, TLR4, TLR5, TLR7, TLR9); an agent that agonizes a nucleicacid sensor (e.g., STING, cGAS, RIG-I (DDX58)); an inflammasomeactivator; a T cell engager (e.g., a multispecific agent thatcross-links CD3 and/or CD28 with a tumor antigen, e.g., a BiTE); acytokine or cytokine variant (e.g., IL-2, IL-7, IL-10, IL-12, IL-15,IL-21, IL-23, IL-27, IL-33, TNF, TL1A, IFNA, IFNB, IFNG); or anycombination thereof.

In some cases, a subject stabilized IL-18 variant polypeptide (e.g., aDR-IL-18 variant, a D2D-IL-18 variant) (e.g., SEQ ID DO: 19) isco-administered with a TGFbeta antagonist such as an anti-TGFbetaantibody; an interferon such as Interferon alpha, Interferon beta, orInterferon gamma; a TNF; TRAIL; a lymphotoxin; LIGHT/TNSF14; and thelike.

In some cases, a subject IL-18 polypeptide (e.g., a stabilized IL-18such as a stabilized DR IL-18 variant or stabilized D2D IL-18 variant,and the like) (e.g., SEQ ID NO: 19) is used in a combination therapy (isco-administered) with an inhibitor of BTLA and/or CD160. In some cases,a subject IL-18 polypeptide (e.g., a stabilized IL-18 such as astabilized DR IL-18 variant or stabilized D2D IL-18 variant, and thelike) (e.g., SEQ ID NO: 19) is used in a combination therapy (isco-administered) with an anti-CD47/SIRPA agent (e.g., anti-CD47,anti-SIRPA, a high affinity CD47 binding agent, a high affinity SIRPAbinding agent, and the like). In some cases, a subject IL-18 polypeptide(e.g., a stabilized IL-18 such as a stabilized DR IL-18 variant orstabilized D2D IL-18 variant, and the like) (e.g., SEQ ID NO: 19) isused in a combination therapy (is co-administered) with an inhibitor ofTIM3 and/or CEACAM1.

In some cases a subject IL-18 polypeptide (e.g., a stabilized IL-18 suchas a stabilized DR IL-18 variant or stabilized D2D IL-18 variant, andthe like) (e.g., SEQ ID NO: 19) is fused to another protein (i.e., a“fusion partner”, a “second polypeptide”). In some embodiments, thesecond polypeptide (the fusion partner for a subject IL-18 variantpolypeptide) specifically binds to a target molecule other than thetarget molecule bound by the IL-18 variant polypeptide portion of thefusion protein (e.g., other than IL-18R for variants that bind IL-18R;or other than IL-18BP for variants that bind to IL-18BP).

Thus, in some embodiments, a subject IL-18 polypeptide (e.g., astabilized IL-18 such as a stabilized DR IL-18 variant or stabilized D2DIL-18 variant, and the like) (e.g., SEQ ID NO:19) is multispecific(e.g., bispecific). The terms “multispecific” or “bispecific” arecommonly used when referring to agents (e.g., ligands or antibodies)that recognize two or more different antigens by virtue of possessing atleast one region (e.g., a ligand or a Fab of a first antibody) that isspecific for a first target, and at least a second region (e.g., aligand or a Fab of a second antibody) that is specific for a secondtarget. A bispecific agent specifically binds to two targets and is thusone type of multispecific agent.

In some embodiments, a subject IL-18 polypeptide (e.g., a stabilizedIL-18 such as a stabilized DR IL-18 variant or stabilized D2D IL-18variant, and the like) (e.g., SEQ ID NO: 19) is multispecific (e.g.,bispecific), such that a first region of the polypeptide includes asubject IL-18 variant polypeptide sequence (i.e., the first regionincludes a IL-18 variant polypeptide), and a second region thatspecifically binds to another target molecule (e.g., an antigen). Forexample, in some cases, a IL-18 polypeptide (e.g., a stabilized IL-18such as a stabilized DR IL-18 variant or stabilized D2D IL-18 variant,and the like) (e.g., SEQ ID NO: 19) is fused to a second polypeptidethat binds specifically to a target molecule other than the targetmolecule bound by the IL-18 variant polypeptide.

Any one of the agents discussed above in the context ofco-administration can be conjugated to a subject IL-18 polypeptide(e.g., a stabilized IL-18 such as a stabilized DR IL-18 variant orstabilized D2D IL-18 variant, and the like) (e.g., SEQ ID NO: 19). Theterm “co-administration” as used herein is meant to encompass suchconjugated compounds. For example, when agent 1 is co-administered withagent 2, the term is meant to encompass embodiments where agent 1 andagent 2 are not conjugated to one another, and is also meant toencompass embodiments where agent 1 and agent 2 are conjugated to oneanother (e.g., where agent 1 and agent 2 are both proteins and agent 1is fused to agent 2).

In some cases, the second region of a multispecific IL-18 polypeptide(e.g., a stabilized IL-18 such as a stabilized DR IL-18 variant orstabilized D2D IL-18 variant, and the like) (e.g., SEQ ID NO: 19)includes a checkpoint inhibitor. In some cases, the second region of amultispecific IL-18 variant polypeptide inhibits one or more proteinsselected from: PD-L1, PD1, CTLA4, TIM3, TIGIT, LAG3, B7H3, B7H4, VISTA,BTLA, CD47, SIRP alpha, CD48, CD155, CD160, TREM2, IDO1, Adenosine 2Areceptor, Aryl hydrocarbon receptor, KIR, and LILRB2.

In some cases, the second region of a multispecific IL-18 polypeptide(e.g., a stabilized IL-18 such as a stabilized DR IL-18 variant orstabilized D2D IL-18 variant, and the like) (e.g., SEQ ID NO: 19)includes an immune agonist. In some cases, the second region of amultispecific IL-18 variant polypeptide agonizes one or more proteinsselected from: ICOS, GITR, 41BB, OX40, and CD40. Additional examples ofimmune agonists that can be used as (or used as part of) a second regionof a multispecific IL-18 polypeptide (e.g., a stabilized IL-18 such as astabilized DR IL-18 variant or stabilized D2D IL-18 variant, and thelike) (e.g., SEQ ID NO: 19) include, but are not limited to: an agent(e.g., antibody or antigen binding region thereof) that agonizes a tumornecrosis factor receptor superfamily (TNFRSF) protein (e.g., GITR, 41BB,OX40, CD27, CD40, HVEM); an agent (e.g., antibody or antigen bindingregion thereof) that agonizes an immunoglobulin superfamily (IgSF)protein (e.g., CD28, ICOS, CD226, NKG2D); an agent that agonizes a TLR(e.g., TLR2, TLR4, TLR5, TLR7, TLR9); an agent that agonizes a nucleicacid sensor (e.g., STING, cGAS, RIG-I (D DX58)); an inflammasomeactivator; a T cell engager (e.g., a multispecific agent thatcross-links CD3 and/or CD28 with a tumor antigen, e.g., a BiTE); acytokine or cytokine variant (e.g., IL-2, IL-7, IL-10, IL-12, IL-15,IL-21, IL-23, IL-27, IL-33, TNF, TL1A, IFNA, IFNB, IFNG); and the like.

In some cases, the second region of a multispecific IL-18 polypeptide(e.g., a stabilized IL-18 such as a stabilized DR IL-18 variant orstabilized D2D IL-18 variant, and the like) (e.g., SEQ ID NO: 19) is acancer cell opsonizing agent. In some cases, the second region of amultispecific IL-18 variant polypeptide targets one or more proteinsselected from: CD19, CD20, CD22, CD24, CD25, CD30, CD33, CD38, CD44,CD47, SIRPA, CD52, CD56, CD70, CD96, CD97, CD99, CD123, CD279 (PD-1),CD274 (PD-L1), EGFR, 17-1A, HER2, CD117, C-Met, PTHR2, and HAVCR2(TIM3). In some cases, the second region of a multispecific IL-18polypeptide (e.g., a stabilized IL-18 such as a stabilized DR IL-18variant or stabilized D2D IL-18 variant, and the like) (e.g., SEQ ID NO:19) is an opsonizing agent that targets one or more proteins selectedfrom: CD19, CD20, CD22, CD24, CD25, CD30, CD33, CD38, CD44, CD47, SIRPA,CD52, CD56, CD70, CD96, CD97, CD99, CD123, CD279 (PD-1), CD274 (PD-L1),EGFR, 17-1A, HER2, CD117, C-Met, PTHR2, and HAVCR2 (TIM3).

For example, in some cases, the second region of a multispecific IL-18polypeptide (e.g., a stabilized IL-18 such as a stabilized DR IL-18variant or stabilized D2D IL-18 variant, and the like) (e.g., SEQ ID NO:19) includes an ectodomain, e.g., an ectodomain from PD-1, PD-L1, CD47(e.g., a high affinity CD47 variant/polypeptide), or SIRPA. (e.g., ahigh affinity SIRPA variant/polypeptide). In some cases, the secondregion of a multispecific IL-18 variant polypeptide specifically bindsan antigen selected from: CTLA-4, Lag-3, BTLA, Tim-3, CD244, CD40,CD40L, CD47, SIRPA, PD-1, and PD-L1.

In some embodiments, the IL-18 variant polypeptide has increasedbiological activity relative to wild type IL-18. In some embodiments,the IL-18 variant comprises no cysteine residues relative to thosenatively occurring in wild type IL-18. In some embodiments, the IL-18variant polypeptide comprises an amino acid sequence with no pegylatedamino acids.

In some embodiments, a subject IL-18 variant polypeptide (e.g., astabilized IL-18) includes a linker (e.g., a linker polypeptide). Forexample, in some embodiments, a subject IL-18 variant polypeptide and afusion partner are separated by a linker (e.g., a linker polypeptide). Alinker polypeptide may have any of a variety of amino acid sequences.Proteins can be joined by a linker polypeptide can be of a flexiblenature (e.g., a flexible linker polypeptide), although other chemicallinkages are not excluded. Suitable linkers include polypeptides ofbetween about 6 amino acids and about 40 amino acids in length, orbetween about 6 amino acids and about 25 amino acids in length. Theselinkers can be produced by using synthetic, linker-encodingoligonucleotides to couple the proteins. Peptide linkers with a degreeof flexibility can be used. The linking peptides may have virtually anyamino acid sequence, bearing in mind that the in some case, linkers willhave a sequence that results in a generally flexible peptide. The use ofsmall amino acids, such as glycine and alanine, are of use in creating aflexible peptide. The creation of such sequences is routine to those ofskill in the art. A variety of different linkers are commerciallyavailable and are considered suitable for use.

In some embodiments the IL-18 variant polypeptide (e.g., a stabilizedIL-18) is co-administered with an immune cell such as atumor-infiltrating lymphocyte (TIL), a CAR-T, TCR-T cell (a T cell withan engineered TCR, e.g., engineered to bind to a target antigen withincreased affinity compared to a natural TCR), CAR-NK cell, or T or NKcell transduced with an engineered T cell receptor. In otherembodiments, the IL-18 variant polypeptide is co-administered with anoncolytic virus.

In some embodiments, a nucleic acid encoding an IL-18 variantpolypeptide (e.g., a stabilized IL-18) is included within an engineered(“altered”) immune cell such as a CAR-T, TCR-T cell, or CAR-NK cell or Tor NK cell transduced with an engineered T cell receptor or aTumor-infiltrating Lymphocyte (TIL). In this instance, the engineeredcell (e.g., altered T cell, altered NK cell, altered TIL) would secretethe IL-18 variant polypeptide. The ability to secrete the IL-18 variantpeptide can be regulated in a contextual manner (e.g., turned on withinthe tumor microenvironment), for instance, by a synthetic NOTCHreceptor.

In some embodiments, a nucleic acid encoding an IL-18 variantpolypeptide (e.g., a stabilized IL-18) is included within an oncolyticvirus. In this instance, cells infected by the oncolytic virus wouldsecrete the IL-18 variant polypeptide.

In some embodiments, an IL-18 variant polypeptide (e.g., a stabilizedIL-18) is administered to a cell, tissue, organ, system, or subject totreat a disease or disorder. In some embodiments, a human IL-18 variantpolypeptide is administered to a cell, tissue, organ, system, orsubject. In some embodiments, a nucleic acid (e.g., DNA, cDNA, mRNA,etc.) encoding at least one human IL-18 variant polypeptide (e.g., astabilized IL-18) is administered to a cell, tissue, organ, system, orsubject.

In some embodiments, a composition of the invention is administered to amurine cell, tissue, organ, system, or subject to treat or prevent adisease or disorder. In some embodiments, a murine IL-18 variantpolypeptide, or a fragment thereof, is administered to a cell, tissue,organ, system, or subject (e.g., a human cell, tissue, organ, system, orsubject). In some embodiments, a nucleic acid (e.g., DNA, cDNA, mRNA,etc.) encoding at least one murine IL-18 variant polypeptide isadministered to a cell, tissue, organ, system, or subject. As notedelsewhere herein, examples of murine IL-18 variant polypeptides (in thiscase DR IL-18 variants) include but are not limited to: SEQ ID NOs:60-72. Also as noted elsewhere herein, examples of murine IL-18 variants(in this case D2D IL-18 variants, which bind to IL-18BP but have reducedbinding to IL-18R) include but are not limited to SEQ ID NOs: 126-190.

In some embodiments, the method comprises administering to a subject,cell, or tissue, an isolated nucleic acid molecule encoding on or moreof the IL-18 variant polypeptides (e.g., a stabilized IL-18) describedherein. Increased level of IL-18 signaling, including by using an IL-18variant polypeptide (e.g., a stabilized IL-18), can be assessed using awide variety of methods, including those disclosed herein, as well asmethods known in the art or to be developed in the future. That is, theroutineer would appreciate, based upon the disclosure provided herein,that increasing the level or activity of IL-18 signaling can be readilyassessed using methods that assess the level of a nucleic acid (e.g.,mRNA) encoding IL-18 or an IL-18 variant polypeptide or fragmentthereof, the level of IL-18 or an IL-18 variant polypeptide or fragmentpolypeptide, and/or the level of IL-18 or an IL-18 variant polypeptideor fragment activity in a biological sample obtained from a subject.

One skilled in the art, based upon the disclosure provided herein, wouldunderstand that the compositions of the disclosure (IL-18 variantpolypeptides such as stabilized IL-18)) is useful in subjects who, inwhole (e.g., systemically) or in part (e.g., locally, cell, tissue,organ), are being or will be, treated for a disease or disorder where anincrease in IL-18 signaling activity would be beneficial. The skilledartisan will appreciate, based upon the teachings provided herein, thatthe diseases and disorders treatable by the compositions and methodsdescribed herein encompass any disease or disorder wherein an increasein IL-18 signaling will promote a positive biologic, physiologic,clinical or therapeutic outcome.

In some embodiments, a method comprises administering to a subject inneed thereof a composition comprising at least one IL-18 polypeptide(e.g., a stabilized IL-18 such as a stabilized DR IL-18 variant orstabilized D2D IL-18 variant, and the like) (e.g., SEQ ID NO: 19), andadministering to the subject a composition comprising an additionalagent. In one such embodiment, the additional agent comprises animmunotherapeutic agent comprising at least one selected from the groupincluding, but not limited to an altered T-cell, a chimeric antigenreceptor T-cell (CAR-T), an armored CAR-T cell, a chimeric antigenreceptor NK-cell (CAR-NK), a TCR-T cell, a Tumor-infiltrating Lymphocyte(TIL), a virus, an antigen, a vaccine, an antibody, an immune checkpointinhibitor, a small molecule, a chemotherapeutic agent, and a stem cell.In some embodiments, a composition comprising at least one IL-18polypeptide (e.g., a stabilized IL-18 such as a stabilized DR IL-18variant or stabilized D2D IL-18 variant, and the like)(e.g., SEQ ID NO:19) is used in a method to increase immune system activity before,during, or after infection by a bacterium, virus, or other pathogen. Insome embodiments, a composition comprising at least one IL-18 variantpolypeptide is used in a method to increase the number and/or activityof immune cells in vitro, in vivo or ex vivo, such as the number and/oractivity of T cells, NK cells, and/or myeloid cells.

In some embodiments, the additional agent comprises an inhibitor of oneor more cytokines. In some embodiments, the inhibitor of one or morecytokines comprises a chemical compound, a protein, a peptide, apeptidomimetic, an antibody, a ribozyme, a small molecule chemicalcompound, or an antisense nucleic acid molecule (e.g., siRNA, miRNA,etc.) that inhibits the expression, activity, or both of one or morecytokines. In some embodiments, the inhibitor inhibits the expression,activity, or both of IL-17, IL-5, or IL-3. In some embodiments, theadministration of a cytokine inhibitor decreases toxicity. In someembodiments, the administration of a cytokine inhibitor increasesefficacy of an administered IL-18 polypeptide (e.g., a stabilized IL-18such as a stabilized DR IL-18 variant or stabilized D2D IL-18 variant,and the like) (e.g., SEQ ID NO: 19).

As used herein, the term “pharmaceutically-acceptable carrier” means achemical composition with which an appropriate IL-18 polypeptide (e.g.,a stabilized IL-18 such as a stabilized DR IL-18 variant or stabilizedD2D IL-18 variant, and the like) (e.g., SEQ ID NO: 19) may be combinedand which, following the combination, can be used to administer theIL-18 polypeptide (e.g., a stabilized IL-18 such as a stabilized DRIL-18 variant or stabilized D2D IL-18 variant, and the like) (e.g., SEQID NO: 19) to a subject.

Production of IL-18 Variants

SUMO Protease

Provided are compositions and methods for making a polypeptide ofinterest (e.g., inactive protein) by using a SUMO (small ubiquitin-likemodifier) protease to cleave a SUMO tag off of a polypeptide ofinterest. The SUMO tag can be place N-terminal to the protein ofinterest and the SUMO protease (e.g., ULP1) can provide for scarlessremoval of the SUMO tag via a cleavage reaction. In some cases, thecleavage will occur in a cell (e.g., in a bacterial cell, in a yeastcell) and in other cases the cleavage will occur cell-free in vitro.

For example, in some cases a fusion protein (that includes a protein ofinterested fused at its N-terminus to a SUMO tag) will be introducedinto a bacterial cell (e.g., via a nucleic acid encoding the fusionprotein) and SUMO protease will also be introduced into the same cell(in such cases, both proteins can be said to be exogenouslyintroduced)—and the cleavage reaction will therefore occur inside of thecell. In some cases, the protease will be encoded by a nucleic acid thatis integrated into the cell's genome and in some cases the nucleic acidwill not be integrated (e.g. can be a plasmid). The SUMO protease can beexpressed from a constitutive promoter or can be operably linked (underthe control of) an inducible promoter such as a rhamnose-induciblepromoter. Any convenient inducible promoter can be used. As such, insome such cases contacting a fusion protein with a SUMO protease in acell (e.g., bacterial cell) can comprise inducing expression of the SUMOprotease.

In some cases, the fusion protein and SUMO protease are encoded by thesame nucleic acid (e.g. plasmid). In some such cases both proteins areoperably linked to the same promoter (e.g., they can be translated froma bicistronic mRNA), and in other cases the two proteins are operablylinked to separate promoters.

In some cases, the fusion protein is contacted with the SUMO protease ina cell-free in vitro environment. For example, a fusion protein can bepurified/isolated and then contacted with a purified SUMO protease(which is readily available).

When the polypeptide of interest is an IL-18 polypeptide (e.g., a wildtype IL-18, a stabilized IL-18 variant, a DR IL-18 variant, a D2D IL-18variant, a stabilized DR IL-18 variant, a stabilized D2D IL-18 variant,and the like) (e.g., SEQ ID NO: 19), such proteins are not fully activewhen the N-terminal amino acid is a methionine (e.g., human wild typeIL-18 protein is normally cleaved by caspase-1 to reveal the N-terminaltyrosine, thus obtaining the active mature form). Thus, methods of thisdisclosure can be used to produce active IL-18 polypeptides. In suchcases, a SUMO (small ubiquitin-like modifier) tag can be used tofacilitate production of large amounts of fusion protein (IL-18 plusSUMO tag), followed by removal of the SUMO tag to reveal an activatedIL-18 protein (e.g., one with a non-methionine, e.g., a tyrosine) at theN-terminus. The SUMO tag can be removed by contacting the fusion proteinwith SUMO protease (e.g., yeast ULP1), which cleaves off the tag toreveal the native N-terminal amino acid (Tyr in the case of humanIL-18). In some cases, the contacting is performed inside of a cell suchas a bacterial cell. In some such cases, the produced active protein canthen be purified, e.g., from a cell lysate (see further details below).

Any convenient SUMO tag can be used—and one such example is set forth asSEQ ID NO: 26. In some cases, e.g., for purposes of subsequentpurification (see below), it is desirable to include an additional tag(e.g., a 6×His tag) on the SUMO tag. SEQ ID NO: 27 provides onenon-limiting example of such a tagged SUMO tag.

Purification Steps

In some embodiments, the subject ‘methods of making’/‘producing’ furtherinclude purifying the polypeptide of interest after the SUMO tag hasbeen removed (e.g., an IL-18 polypeptide such as a stabilized IL-18variant, a DR IL-18 variant, a D2D IL-18 variant, a stabilized DR IL-18variant, a stabilized D2D IL-18 variant, and the like) (e.g., SEQ ID NO:19). In some such cases the cleavage (SUMO tag removal) occurs cell-freein vitro and the purification can include removal of the SUMO tag andSUMO protease. For example, in some cases the SUMO protease and/or theSUMO tag can be tagged (e.g., His tagged), which can in some casesfacilitate removal of these components after cleavage, e.g., forpurification of the polypeptide of interest, e.g., using immobilizedmetal affinity chromatography (IMAC) such as a nickel column.

In some cases, the cleavage (SUMO tag removal) occurs in a cell (e.g., abacterial cell as discussed above) and the polypeptide of interested ispurified from a cell lysate. In some cases, the polypeptide ofinterested is secreted into the medium (e.g., in some cases when usingyeast cells)—and is then purified from the medium. Any convenient stepor series of steps can be used to purify proteins from, e.g., bacteriallysates or media into which proteins have been secreted. In some cases(e.g., in some cases in which the polypeptide of interest is an IL-18polypeptide such as a stabilized IL-18 variant, a DR IL-18 variant, aD2D IL-18 variant, a stabilized DR IL-18 variant, a stabilized D2D IL-18variant, and the like) (e.g., SEQ ID NO: 19), purification includesion-exchange chromatography (e.g., Capto Q), IMAC (e.g., to remove anyresidual his-tagged SUMO, uncleaved protein, and/or protease),multimodal chromatography (e.g., Capto MMC chromatography-Capto MMC is amultimodal salt-tolerant resin for capture and intermediate purificationof proteins from large feed volumes by packed bed chromatography) andhydrophobic interaction chromatography (HIC) (e.g., in some cases phenylsepharose HIC). In some cases, high-performance Q chromatography is alsoperformed. In some such cases, ultrafiltration (UF) and diafiltration(DF) steps are performed prior to each chromatography step. As anexample of one possible purification process, see FIG. 48 .

It will be appreciated by one of skill in the art, when armed with thepresent disclosure including the methods detailed herein, that theinvention is not limited to treatment of a disease or disorder once itis established. Particularly, the symptoms of the disease or disorderneed not have manifested to the point of detriment to the subject;indeed, the disease or disorder need not be detected in a subject beforetreatment is administered. That is, significant pathology from diseaseor disorder does not have to occur before the present invention mayprovide benefit. Therefore, the present invention, as described morefully herein, includes a method for preventing diseases and disorders ina subject, in that an activator of IL-18 activity as discussed elsewhereherein, can be administered to a subject prior to the onset of thedisease or disorder, thereby preventing the disease or disorder fromdeveloping.

Pharmaceutical Compositions of the Disclosure

Compositions comprising a polypeptide, a polypeptide fragment, an IL-18polypeptide (e.g., a stabilized IL-18 such as a stabilized DR IL-18variant or stabilized D2D IL-18 variant, and the like) (e.g., SEQ ID NO:19) as described elsewhere herein can be formulated and administered toa subject, as now described. By way of non-limiting examples, a IL-18polypeptide (e.g., a stabilized IL-18 such as a stabilized DR IL-18variant or stabilized D2D IL-18 variant, and the like) (e.g., SEQ ID NO:19) for the treatment and/or prevention of a disease or disorder can beformulated and administered to a subject, in any convenient way, e.g.,as now described.

The disclosure encompasses the preparation and use of pharmaceuticalcompositions comprising a composition useful for the treatment orprevention of a disease or disorder, disclosed herein as an activeingredient (an IL-18 polypeptide, e.g., a stabilized IL-18 such as astabilized DR IL-18 variant or stabilized D2D IL-18 variant, and thelike) (e.g., SEQ ID NO: 19). Such a pharmaceutical composition mayconsist of the active ingredient alone, in a form suitable foradministration to a subject, or the pharmaceutical composition maycomprise the active ingredient and one or more pharmaceuticallyacceptable carriers, one or more additional ingredients, or somecombination of these. The active ingredient may be present in thepharmaceutical composition in the form of a physiologically acceptableester or salt, such as in combination with a physiologically acceptablecation or anion, as is well known in the art.

As used herein, the term “pharmaceutically-acceptable carrier” means achemical composition with which an appropriate IL-18 polypeptide (e.g.,a stabilized IL-18 such as a stabilized DR IL-18 variant or stabilizedD2D IL-18 variant, and the like) (e.g., SEQ ID NO: 19), may be combinedand which, following the combination, can be used to administer to asubject.

In some embodiments, pharmaceutical compositions can include large,slowly metabolized macromolecules such as proteins, polysaccharides suchas chitosan, polylactic acids, polyglycolic acids and copolymers (suchas latex functionalized Sepharose™, agarose, cellulose, and the like),polymeric amino acids, amino acid copolymers, and lipid aggregates (suchas oil droplets or liposomes).

The pharmaceutical compositions useful for practicing the subjectmethods (e.g., the IL-18 polypeptide such as a wild type IL-18, astabilized IL-18, a DR IL-18, a D2D IL-18, a stabilized DR IL-18, astabilized D2D IL-18, and the like) (e.g., SEQ ID NO: 19) may beadministered to deliver a dose of between about 0.1 ng and 100 mg per kg(e.g., 1 ng to 100 mg, 100 ng to 100 mg, 1 ug to 100 mg, 100 ug to 100mg, 1 mg to 100 mg, 25 mg to 100 mg, 50 mg to 100 mg, 1 ng to 80 mg, 100ng to 80 mg, 1 ug to 80, 100 ug to 80 mg, 1 mg to 80 mg, 25 mg to 80 mg,50 mg to 80 mg, 1 ng to 50 mg, 100 ng to 50 mg, 1 ug to 50 mg, 100 ug to50 mg, 1 mg to 50 mg, 25 mg to 50 mg, 50 mg to 50 mg, 1 ng to 50 mg, 100ng to 50 mg, 1 ug to 50 mg, 100 ug to 50 mg, 1 mg to 50 mg, or 25 mg to50 mg per kg). Dosing is described in more detail elsewhere herein.

In various embodiments, the pharmaceutical compositions useful in themethods of the invention may be administered, by way of example,systemically, parenterally, subcutaneously, intraperitoneally, ortopically, such as, in oral formulations, inhaled formulations,including solid or aerosol, and by topical or other similarformulations, or intraocularly. In addition to the appropriatetherapeutic composition, such pharmaceutical compositions may containpharmaceutically acceptable carriers and other ingredients known toenhance and facilitate drug administration. Other possible formulations,such as nanoparticles, liposomes, other preparations containing theactive ingredient, and immunologically based systems may also be used toadminister an appropriate modulator thereof, according to the methods ofthe invention.

A carrier may bear a subject agent (e.g., IL-18 variant polypeptide) ina variety of ways, including covalent bonding either directly or via alinker group, and non-covalent associations. Suitable covalent-bondcarriers include proteins such as albumins, peptides, andpolysaccharides such as aminodextran, each of which have multiple sitesfor the attachment of moieties. A carrier may also bear a IL-18polypeptide (e.g., a stabilized IL-18 such as a stabilized DR IL-18variant or stabilized D2D IL-18 variant, and the like) (e.g., SEQ ID NO:19) by non-covalent associations, such as non-covalent bonding or byencapsulation. The nature of the carrier can be either soluble orinsoluble for purposes of the invention.

Acceptable carriers, excipients, or stabilizers are non-toxic torecipients at the dosages and concentrations employed, and includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyidimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).Formulations to be used for in vivo administration must be sterile. Thisis readily accomplished by filtration through sterile filtrationmembranes.

The active ingredients may also be entrapped in microcapsule prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsule and poly-(methylmethacylate) microcapsule,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

Compositions can be prepared as injectables, either as liquid solutionsor suspensions; solid forms suitable for solution in, or suspension in,liquid vehicles prior to injection can also be prepared. The preparationalso can be emulsified or encapsulated in liposomes or micro particlessuch as polylactide, polyglycolide, or copolymer for enhanced adjuvanteffect, as discussed above. Langer, Science 249: 1527, 1990 and Hanes,Advanced Drug Delivery Reviews 28: 97119, 1997. The agents of thisinvention can be administered in the form of a depot injection orimplant preparation which can be formulated in such a manner as topermit a sustained or pulsatile release of the active ingredient. Thepharmaceutical compositions are generally formulated as sterile,substantially isotonic and in full compliance with all GoodManufacturing Practice (GMP) regulations of the U.S. Food and DrugAdministration.

As used herein, the term “physiologically acceptable” ester or saltmeans an ester or salt form of the active ingredient which is compatiblewith any other ingredients of the pharmaceutical composition, which isnot deleterious to the subject to which the composition is to beadministered.

The formulations of the pharmaceutical compositions described herein maybe prepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofbringing the active ingredient into association with a carrier or one ormore other accessory ingredients, and then, if necessary or desirable,shaping or packaging the product into a desired single- or multi-doseunit.

Although the descriptions of pharmaceutical compositions provided hereinare principally directed to pharmaceutical compositions which aresuitable for ethical administration to humans, it will be understood bythe skilled artisan that such compositions are generally suitable foradministration to animals of all sorts. Modification of pharmaceuticalcompositions suitable for administration to humans in order to renderthe compositions suitable for administration to various animals is wellunderstood, and the ordinarily skilled veterinary pharmacologist candesign and perform such modification with merely ordinary, if any,experimentation.

Pharmaceutical compositions that are useful in the methods of theinvention may be prepared, packaged, or sold in formulations suitablefor oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal,buccal, intravenous, transdermal, intralesional, subcutaneous,intramuscular, ophthalmic, intrathecal and other known routes ofadministration. Other contemplated formulations include projectednanoparticles, liposomal preparations, other preparations containing theactive ingredient, and immunologically-based formulations.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in bulk, as a single unit dose, or as a plurality of single unitdoses. As used herein, a “unit dose” is discrete amount of thepharmaceutical composition comprising a predetermined amount of theactive ingredient. The amount of the active ingredient is generallyequal to the dosage of the active ingredient which would be administeredto a subject or a convenient fraction of such a dosage such as, forexample, one-half or one-third of such a dosage.

The relative amounts of the active ingredient, the pharmaceuticallyacceptable carrier, and any additional ingredients in a pharmaceuticalcomposition of the invention will vary, depending upon the identity,size, and condition of the subject treated and further depending uponthe route by which the composition is to be administered. By way ofexample, the composition may comprise between 0.1% and 100% (w/w) activeingredient.

In addition to the active ingredient, a pharmaceutical composition ofthe invention may further comprise one or more additionalpharmaceutically active agents.

Controlled- or sustained-release formulations of a pharmaceuticalcomposition of the invention may be made using conventional technology.

A formulation of a pharmaceutical composition of the invention suitablefor oral administration may be prepared, packaged, or sold in the formof a discrete solid dose unit including, but not limited to, a tablet, ahard or soft capsule, a cachet, a troche, or a lozenge, each containinga predetermined amount of the active ingredient. Other formulationssuitable for oral administration include, but are not limited to, apowdered or granular formulation, an aqueous or oily suspension, anaqueous or oily solution, or an emulsion.

Pharmaceutically acceptable excipients used in the manufacture ofpharmaceutical compositions include, but are not limited to, inertdiluents, granulating and disintegrating agents, binding agents, andlubricating agents. Known dispersing agents include, but are not limitedto, potato starch and sodium starch glycollate. Known surface activeagents include, but are not limited to, sodium lauryl sulphate. Knowndiluents include, but are not limited to, calcium carbonate, sodiumcarbonate, lactose, microcrystalline cellulose, calcium phosphate,calcium hydrogen phosphate, and sodium phosphate. Known granulating anddisintegrating agents include, but are not limited to, corn starch andalginic acid. Known binding agents include, but are not limited to,gelatin, acacia, pre-gelatinized maize starch, polyvinylpyrrolidone, andhydroxypropyl methylcellulose. Known lubricating agents include, but arenot limited to, magnesium stearate, stearic acid, silica, and talc.

Liquid formulations of a pharmaceutical composition of the invention maybe prepared, packaged, and sold either in liquid form or in the form ofa dry product intended for reconstitution with water or another suitablevehicle prior to use.

Liquid suspensions may be prepared using conventional methods to achievesuspension of the active ingredient in an aqueous or oily vehicle.Aqueous vehicles include, for example, water and isotonic saline. Oilyvehicles include, for example, almond oil, oily esters, ethyl alcohol,vegetable oils such as arachis, olive, sesame, or coconut oil,fractionated vegetable oils, and mineral oils such as liquid paraffin.Liquid suspensions may further comprise one or more additionalingredients including, but not limited to, suspending agents, dispersingor wetting agents, emulsifying agents, demulcents, preservatives,buffers, salts, flavorings, coloring agents, and sweetening agents. Oilysuspensions may further comprise a thickening agent.

Known suspending agents include, but are not limited to, sorbitol syrup,hydrogenated edible fats, sodium alginate, polyvinylpyrrolidone, gumtragacanth, gum acacia, and cellulose derivatives such as sodiumcarboxymethylcellulose, methylcellulose, and 15hydroxypropylmethylcellulose. Known dispersing or wetting agentsinclude, but are not limited to, naturally-occurring phosphatides suchas lecithin, condensation products of an alkylene oxide with a fattyacid, with a long chain aliphatic alcohol, with a partial ester derivedfrom a fatty acid and a hexitol, or with a partial ester derived from afatty acid and a hexitol anhydride (e.g. polyoxyethylene stearate,heptadecaethyleneoxycetanol, polyoxyethylene sorbitol monooleate andpolyoxyethylene sorbitan monooleate, respectively). Known emulsifyingagents include, but are not limited to, lecithin and acacia. Knownpreservatives include, but are not limited to, methyl, ethyl, orn-propyl-para-hydroxybenzoates, ascorbic acid, and sorbic acid. Knownsweetening agents include, for example, glycerol, propylene glycol,sorbitol, sucrose, and saccharin. Known thickening agents for oilysuspensions include, for example, beeswax, hard paraffin, and cetylalcohol.

Liquid solutions of the active ingredient in aqueous or oily solventsmay be prepared in substantially the same manner as liquid suspensions,the primary difference being that the active ingredient is dissolved,rather than suspended in the solvent. Liquid solutions of thepharmaceutical composition of the invention may comprise each of thecomponents described with regard to liquid suspensions, it beingunderstood that suspending agents will not necessarily aid dissolutionof the active ingredient in the solvent. Aqueous solvents include, forexample, water and isotonic saline. Oily solvents include, for example,almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis,olive, sesame, or coconut oil, fractionated vegetable oils, and mineraloils such as liquid paraffin.

Powdered and granular formulations of a pharmaceutical preparation ofthe invention may be prepared using known methods. Such formulations maybe administered directly to a subject, used, for example, to formtablets, to fill capsules, or to prepare an aqueous or oily suspensionor solution by addition of an aqueous or oily vehicle thereto. Each ofthese formulations may further comprise one or more of dispersing orwetting agent, a suspending agent, and a preservative. Additionalexcipients, such as fillers and sweetening, flavoring, or coloringagents, may also be included in these formulations.

A pharmaceutical composition of the invention may also be prepared,packaged, or sold in the form of oil-in-water emulsion or a water-in-oilemulsion. The oily phase may be a vegetable oil such as olive or arachisoil, a mineral oil such as liquid paraffin, or a combination of these.Such compositions may further comprise one or more emulsifying agentssuch as naturally occurring gums such as gum acacia or gum tragacanth,naturally-occurring phosphatides such as soybean or lecithinphosphatide, esters or partial esters derived from combinations of fattyacids and hexitol anhydrides such as sorbitan monooleate, andcondensation products of such partial esters with ethylene oxide such aspolyoxyethylene sorbitan monooleate. These emulsions may also containadditional ingredients including, for example, sweetening or flavoringagents.

Methods for impregnating or coating a material with a chemicalcomposition are known in the art, and include, but are not limited tomethods of depositing or binding a chemical composition onto a surface,methods of incorporating a chemical composition into the structure of amaterial during the synthesis of the material (i.e., such as with aphysiologically degradable material), and methods of absorbing anaqueous or oily solution or suspension into an absorbent material, withor without subsequent drying.

As used herein, “parenteral administration” of a pharmaceuticalcomposition includes any route of administration characterized byphysical breaching of a tissue of a subject and administration of thepharmaceutical composition through the breach in the tissue. Parenteraladministration thus includes, but is not limited to, administration of apharmaceutical composition by injection of the composition, byapplication of the composition through a surgical incision, byapplication of the composition through a tissue-penetrating non-surgicalwound, and the like. In particular, parenteral administration iscontemplated to include, but is not limited to, cutaneous, subcutaneous,intraperitoneal, intravenous, intramuscular, intracisternal injection,and kidney dialytic infusion techniques. In some cases, a subject IL-18polypeptide (e.g., a stabilized IL-18 such as a stabilized DR IL-18variant or stabilized D2D IL-18 variant, and the like) (e.g., SEQ ID NO:19) is administered subcutaneously.

Formulations of a pharmaceutical composition suitable for parenteraladministration comprise the active ingredient combined with apharmaceutically acceptable carrier, such as sterile water or sterileisotonic saline. Such formulations may be prepared, packaged, or sold ina form suitable for bolus administration or for continuousadministration. Injectable formulations may be prepared, packaged, orsold in unit dosage form, such as in ampules or in multi-dose containerscontaining a preservative. Formulations for parenteral administrationinclude, but are not limited to, suspensions, solutions, emulsions inoily or aqueous vehicles, pastes, and implantable sustained-release orbiodegradable formulations. Such formulations may further comprise oneor more additional ingredients including, but not limited to,suspending, stabilizing, or dispersing agents. In some embodiments of aformulation for parenteral administration, the active ingredient isprovided in dry (i.e., powder or granular) form for reconstitution witha suitable vehicle (e.g., sterile pyrogen-free water) prior toparenteral administration of the reconstituted composition.

The pharmaceutical compositions may be prepared, packaged, or sold inthe form of a sterile injectable aqueous or oily suspension or solution.This suspension or solution may be formulated according to the knownart, and may comprise, in addition to the active ingredient, additionalingredients such as the dispersing agents, wetting agents, or suspendingagents described herein. Such sterile injectable formulations may beprepared using a non-toxic parenterally-acceptable diluent or solvent,such as water or 1,3-butane diol, for example. Other acceptable diluentsand solvents include, but are not limited to, Ringer's solution,isotonic sodium chloride solution, and fixed oils such as syntheticmono- or di-glycerides. Other parentally-administrable formulationswhich are useful include those which comprise the active ingredient inmicrocrystalline form, in a liposomal preparation, or as a component ofa biodegradable polymer systems. Compositions for sustained release orimplantation may comprise pharmaceutically acceptable polymeric orhydrophobic materials such as an emulsion, an ion exchange resin, asparingly soluble polymer, or a sparingly soluble salt.

Formulations suitable for topical administration include, but are notlimited to, liquid or semi-liquid preparations such as liniments,lotions, oil-in-water or water-in-oil emulsions such as creams,ointments or pastes, and solutions or suspensions.Topically-administrable formulations may, for example, comprise fromabout 1% to about 10% (w/w) active ingredient, although theconcentration of the active ingredient may be as high as the solubilitylimit of the active ingredient in the solvent. Formulations for topicaladministration may further comprise one or more of the additionalingredients described herein.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for pulmonary administration via thebuccal cavity. Such a formulation may comprise dry particles whichcomprise the active ingredient and which have a diameter in the rangefrom about 0.5 to about 7 nanometers, and preferably from about 1 toabout 6 nanometers. Such compositions are conveniently in the form ofdry powders for administration using a device comprising a dry powderreservoir to which a stream of propellant may be directed to dispersethe powder or using a self-propelling solvent/powder-dispensingcontainer such as a device comprising the active ingredient dissolved orsuspended in a low-boiling propellant in a sealed container. Preferably,such powders comprise particles wherein at least 98% of the particles byweight have a diameter greater than 0.5 nanometers and at least 95% ofthe particles by number have a diameter less than 7 nanometers. Morepreferably, at least 95% of the particles by weight have a diametergreater than 1 nanometer and at least 90% of the particles by numberhave a diameter less than 6 nanometers. Dry powder compositionspreferably include a solid fine powder diluent such as sugar and areconveniently provided in a unit dose form.

Low boiling propellants generally include liquid propellants having aboiling point of below 65° F. at atmospheric pressure. Generally, thepropellant may constitute 50 to 99.9% (w/w) of the composition, and theactive ingredient may constitute 0.1 to 20% (w/w) of the composition.The propellant may further comprise additional ingredients such as aliquid non-ionic or solid anionic surfactant or a solid diluent(preferably having a particle size of the same order as particlescomprising the active ingredient).

Pharmaceutical compositions of the invention formulated for pulmonarydelivery may also provide the active ingredient in the form of dropletsof a solution or suspension. Such formulations may be prepared,packaged, or sold as aqueous or dilute alcoholic solutions orsuspensions, optionally sterile, comprising the active ingredient, andmay conveniently be administered using any nebulization or atomizationdevice. Such formulations may further comprise one or more additionalingredients including, but not limited to, a flavoring agent such assaccharin sodium, a volatile oil, a buffering agent, a surface activeagent, or a preservative such as methylhydroxybenzoate. The dropletsprovided by this route of administration preferably have an averagediameter in the range from about 0.1 to about 200 nanometers. Theformulations described herein as being useful for pulmonary delivery arealso useful for intranasal delivery of a pharmaceutical composition ofthe invention. Another formulation suitable for intranasaladministration is a coarse powder comprising the active ingredient andhaving an average particle from about 0.2 to 500 micrometers.

Such a formulation is administered in the manner in which snuff is takeni.e. by rapid inhalation through the nasal passage from a container ofthe powder held close to the nares. Formulations suitable for nasaladministration may, for example, comprise from about as little as 0.1%(w/w) and as much as 100% (w/w) of the active ingredient, and mayfurther comprise one or more of the additional ingredients describedherein.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for buccal administration. Suchformulations may, for example, be in the form of tablets or lozengesmade using conventional methods, and may, for example, contain 0.1 to20% (w/w) active ingredient, the balance comprising an orallydissolvable or degradable composition and, optionally, one or more ofthe additional ingredients described herein. Alternately, formulationssuitable for buccal administration may comprise a powder or anaerosolized or atomized solution or suspension comprising the activeingredient. Such powdered, aerosolized, or aerosolized formulations,when dispersed, preferably have an average particle or droplet size inthe range from about 0.1 to about 200 nanometers, and may furthercomprise one or more of the additional ingredients described herein.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for ophthalmic administration. Suchformulations may, for example, be in the form of eye drops including,for example, a 0.1-1.0% (w/w) solution or suspension of the activeingredient in an aqueous or oily liquid carrier. Such drops may furthercomprise buffering agents, salts, or one or more other of the additionalingredients described herein. Other ophthalmically-administrableformulations which are useful include those which comprise the activeingredient in microcrystalline form or in a liposomal preparation.

As used herein, “additional ingredients” include, but are not limitedto, one or more of the following: excipients; surface active agents;dispersing agents; inert diluents; granulating and disintegratingagents; binding agents; lubricating agents; sweetening agents; flavoringagents; coloring agents; preservatives; physiologically degradablecompositions such as gelatin; aqueous vehicles and solvents; oilyvehicles and solvents; suspending agents; dispersing or wetting agents;emulsifying agents, demulcents; buffers; salts; thickening agents;fillers; emulsifying agents; antioxidants; antibiotics; antifungalagents; stabilizing agents; and pharmaceutically acceptable polymeric orhydrophobic materials. Other “additional ingredients” which may beincluded in the pharmaceutical compositions of the invention are knownin the art and described, for example in Genaro, ed., 1985, Remington'sPharmaceutical Sciences, Mack Publishing Co., Easton, Pa., which isincorporated herein by reference.

Example Formulations

In some embodiments, the formulation comprises an active ingredientcomprising an IL-18 polypeptide, one or more of a buffering agent tocontrol pH, one or more of a stabilizer, one or more of a solvent, asurfactant, a reagent for preventing oxidation, and a stabilizer. Insome embodiments, formulations of a IL-18 polypeptide comprise one ormore of: L-Histidine (His), L-Histidine Hydrochloride (His-HCl),sucrose, Polysorbate 80, L-Methionine and disodium salt of EDTA.

One example of an appropriate formulation for a IL-18 polypeptide (e.g.,a stabilized IL-18 such as a stabilized DR IL-18 variant or stabilizedD2D IL-18 variant, and the like) (e.g., SEQ ID NO: 19) that was arrivedat via experimentation includes: (1) an IL-18 polypeptide (e.g., a wildtype IL-18, a stabilized IL-18, a DR IL-18, a D2D IL-18, a stabilized DRIL-18, a stabilized D2D IL-18, and the like) (e.g., SEQ ID NO: 19); (2)a buffering agent to maintain to pH between 6.2 and 6.8 (e.g., in somecases 6.5)(e.g., His/His-HCl at 8-12 mM, e.g., at 10 mM); (3) a sugar(e.g., sucrose, e.g., at 6-10%; in some cases 8%); (4) a chelating agent(e.g., EDTA, e.g., at 0.05-1.5 mM, e.g., in some cases 0.1 mM); (5) anagent to prevent oxidation (e.g., L-methionine, e.g., at 4-6 mM, e.g.,in some cases 5 mM), and (6) an agent to prevent protein adsorption(e.g., PS80, e.g., at 0.01-0.03% w/v, e.g., 0.02%).

As such, in some cases a formulation includes: (1) an IL-18 polypeptide(e.g., a wild type IL-18, a stabilized IL-18, a DR IL-18, a D2D IL-18, astabilized DR IL-18, a stabilized D2D IL-18, and the like) (e.g., SEQ IDNO: 19); (2) about 10 mM His/His-HCl; (3) about 8% sucrose; (4) about0.1 mM EDTA; (5) about 5 mM L-Methionine; and (6) about 0.02% (w/v)PS80.

In some of the above embodiments (e.g., if the formulation is to beadministered subcutaneously), the IL-18 polypeptide (e.g., a wild typeIL-18, a stabilized IL-18, a DR IL-18, a D2D IL-18, a stabilized DRIL-18, a stabilized D2D IL-18, and the like) (e.g., SEQ ID NO: 19) willbe present at a concentration in a range of from 10-100 mg/ml (e.g.,10-90, 10-75, 10-60, 10-50, 10-40, 10-30, 25-100, 25-90, 25-75, 25-60,25-50, 25-40, 25-30, 30-100, 30-90, 30-75, 30-60, 30-50, 30-40, 40-100,40-90, 40-75, 40-60, 40-50, 50-100, 50-90, 50-75, or 50-60 mg/ml). Insome of the above embodiments (e.g., if the formulation is to beadministered subcutaneously), the IL-18 polypeptide (e.g., a wild typeIL-18, a stabilized IL-18, a DR IL-18, a D2D IL-18, a stabilized DRIL-18, a stabilized D2D IL-18, and the like) (e.g., SEQ ID NO: 19) willbe present at a concentration in a, or 50-60 mg/ml). In some of theabove embodiments (e.g., if the formulation is to be administeredsubcutaneously), the IL-18 polypeptide (e.g., a wild type IL-18, astabilized IL-18, a DR IL-18, a D2D IL-18, a stabilized DR IL-18, astabilized D2D IL-18, and the like) (e.g., SEQ ID NO: 19) will bepresent at a concentration in a range of from 20-30 mg/ml.

In some embodiments, the drug product is formulated in batches. In someembodiments, batches are 1.5 L. In other embodiments, batches are 10.5L. Batches may include, for example, the IL-18 polypeptide of SEQ ID NO:19, L-Histidine, L-Histidine Hydrochloride, sucrose, polysorbate 80,L-Methionine, disodium salt of EDTA, and water for injections. In someembodiments, a batch comprises between 40 and 50 g SEQ ID NO: 19,between 1.00 and 2.00 g of L-Histidine; between 0.25 and 1.00 gL-Histidine Hydrochloride, between 100.00 and 150.00 g of sucrose;between 0.01 and 0.60 g polysorbate 80; between 1 and 1.5 gL-Methionine; between 0.005 and 0.1 g disodium salt of EDTA; and aquantity sufficient to about 1.5 L of water. In some embodiments, abatch comprises about 45 g SEQ ID NO: 19, about 1.91 g of L-Histidine;about 0.57 g L-Histidine Hydrochloride, about 120.0 g of sucrose; about0.30 g polysorbate 80; about 1.13 g L-Methionine; about 0.06 g disodiumsalt of EDTA; and a quantity sufficient to about 1.5 L of water. In someembodiments, a 10.5 L batch comprises between 300 and 350 g SEQ ID NO:19, between 10.00 and 15.00 g of L-Histidine; between 3.50 and 4.50 gL-Histidine Hydrochloride, between 800.00 and 900.00 g of sucrose;between 1.5 and 2.5 g polysorbate 80; between 7.00 and 8.00 gL-Methionine; between 0.1 and 0.6 g disodium salt of EDTA; and aquantity sufficient to about 10.5 L of water. In some embodiments, a10.5 L batch comprises about 315 g SEQ ID NO: 19, about 13.34 g ofL-Histidine; about 3.99 g L-Histidine Hydrochloride, about 840.0 g ofsucrose; about 2.1 g polysorbate 80; about 7.88 g L-Methionine; about0.42 g disodium salt of EDTA; and a quantity sufficient to about 1.5 Lof water.

A formulation of the disclosure can be maintained at a pH, for example,to help stabilize or maintain activity of a compound or polypeptidedisclosed herein. The pH of the disclosed formulation can range, forexample, from about 3 to about 12. The pH of the composition can be, forexample, from about 3 to about 4, from about 4 to about 5, from about 5to about 6, from about 6 to about 7, from about 7 to about 8, from about8 to about 9, from about 9 to about 10, from about 10 to about 11, orfrom about 11 to about 12 pH units. The pH of the composition can be,for example, about 3, about 4, about 5, about 6, about 7, about 8, about9, about 10, about 11, or about 12 pH units. The pH of the compositioncan be, for example, at least 3, at least 4, at least 5, at least 6, atleast 7, at least 8, or at least 9 pH units. The pH of the compositioncan be, for example, at most 5, at most 6, at most 7, at most 8, at most9, at most 10, at most 11, or at most 12 pH units.

A formulation disclosed herein can have a pH of from about 5.5 to about8. For example, a formulation of the present disclosure can have a pH ofabout 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7,about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, or about8.0. In some embodiments, the pH is 6.5±0.6, 6.5±0.5, 6.5±0.4, 6.5±0.3,6.5±0.2, 6.5±0.1, about 6.5, or 6.5. In some embodiments, the pH is6.2±0.6, 6.2±0.5, 6.2±0.4, 6.2±0.3, 6.2±0.2, 6.2±0.1, about 6.2, or 6.2.In some embodiments, the pH is 6.8±0.6, 6.8±0.5, 6.8±0.4, 6.8±0.3,6.8±0.2, 6.8±0.1, about 6.8, or 6.8. In some embodiments, the pH is7.4±0.6, 7.4±0.5, 7.4±0.4, 7.4±0.3, 7.4±0.2, 7.4±0.1, about 7.4, or 7.4.In some embodiments, the pH is about 6.2 to about 6.8. If the pH isoutside the range desired by the formulator, the pH can be adjusted byusing sufficient pharmaceutically-acceptable acids and bases.

In some embodiments, a formulation disclosed herein can comprise abuffering agent. In some embodiments, the buffering agent serves tomaintain a stable pH and to help stabilize a compound or polypeptidedisclosed herein. In some embodiments, a buffer system comprises atleast one buffering agent that has a buffering range that overlaps fullyor in part the range of pH 5.5-7.4. In some embodiments, the buffer hasa pKa of about 6.5±0.5, or 6.2±0.5.

Dosing

Typically dosages of an IL-18 polypeptide (e.g., a wild type IL-18, astabilized IL-18 variant, a DR IL-18 variant, a D2D IL-18 variant, astabilized DR IL-18 variant, a stabilized D2D IL-18 variant, and thelike) (e.g., SEQ ID NO: 19) which may be administered to an animal,preferably a human, range in amount from 0.001 mg to about 10 mg perkilogram (mpk) of body weight of the animal. In some embodiments, anIL-18 polypeptide (e.g., a wild type IL-18, a stabilized IL-18 variant,a DR IL-18 variant, a D2D IL-18 variant, a stabilized DR IL-18 variant,a stabilized D2D IL-18 variant, and the like) (e.g., SEQ ID NO: 19) isadministered to an individual at a dose in a range of from 0.01-4 mpk(e.g., 0.01-3.5, 0.01-3.3, 0.01-3, 0.01-1.5, 0.01-2, 0.01-1.5, 0.01-1,0.01-0.5, 0.01-0.35, 0.025-4, 0.025-3.5, 0.025-3.3, 0.025-3, 0.025-1.5,0.025-2, 0.025-1.5, 0.025-1, 0.0250.5, 0.025-0.35, 0.05-4, 0.05-3.5,0.05-3.3, 0.05-3, 0.05-1.5, 0.05-2, 0.05-1.5, 0.05-1, 0.05-0.5,0.05-0.35, 0.1-4, 0.1-3.5, 0.1-3.3, 0.1-3, 0.1-1.5, 0.1-2, 0.1-1.5,0.1-1, 0.1-0.5, 0.1-0.35, 0.25-4, 0.25-3.5, 0.25-3.3, 0.25-3, 0.25-1.5,0.25-2, 0.25-1.5, 0.25-1, 0.25-0.5, 0.25-0.35, 0.5-4, 0.5-3.5, 0.5-3.3,0.5-3, 0.5-1.5, 0.5-2, 0.5-1.5, or 0.5-1 mpk). In some embodiments, anIL-18 polypeptide (e.g., a wild type IL-18, a stabilized IL-18 variant,a DR IL-18 variant, a D2D IL-18 variant, a stabilized DR IL-18 variant,a stabilized D2D IL-18 variant, and the like) (e.g., SEQ ID NO: 19) isadministered to an individual at a dose in a range of from 0.05-3.5 mpk(e.g., 0.05-3.3, 0.05-3, 0.05-1.5, 0.05-2, 0.05-1.5, 0.05-1, 0.05-0.5,0.05-0.35, 0.1-3.5, 0.1-3.3, 0.1-3, 0.1-1.5, 0.1-2, 0.1-1.5, 0.1-1,0.1-0.5, 0.1-0.35, 0.25-3.5, 0.25-3.3, 0.25-3, 0.25-1.5, 0.25-2,0.25-1.5, 0.25-1, 0.25-0.5, 0.25-0.35, 0.5-3.5, 0.5-3.3, 0.5-3, 0.5-1.5,0.5-2, 0.5-1.5, or 0.5-1 mpk). In some cases the compound can beadministered to an animal as frequently as several times daily, or itcan be administered less frequently, such as once a day, once a week,once every two weeks, once a month, or even less frequently, such asonce every several months or even once a year or less.

However, as noted above, in some cases it is delivered once per week orless.

Methods of Administration

Frequency of Administration

One of skill in the art will appreciate that the IL-18 proteinsdiscussed herein can be administered acutely (e.g., over a short period)or chronically (e.g., over a long period of time, such as several monthsor a year or more). One of skill in the art will appreciate that theycan be administered singly or in any combination with other agents.Further, the IL-18 proteins described herein can be administered singlyor in any combination in a temporal sense, in that they may beadministered concurrently, and/or before, and/or after each other. Oneof ordinary skill in the art will appreciate, based on the disclosureprovided herein, that IL-18 protein compositions can be used to treat adisease or disorder in a subject in need thereof, and that such proteinscan be used alone or in any combination with another agent to affect atherapeutic result.

As surprisingly demonstrated in the working examples below,administration of an IL-18 polypeptide (e.g., a wild type IL-18, astabilized IL-18 variant, a DR IL-18 variant, a D2D IL-18 variant, astabilized DR IL-18 variant, a stabilized D2D IL-18 variant, and thelike) (e.g., SEQ ID NO: 19) can in some cases be detrimental (e.g.,toxic—can cause anemia) if it is administered too frequently, e.g., morethan once per week. For example, the working examples below demonstratedthat administrations of DR-18 of twice per week in primates (monkeys)had a dose-dependent reduction in hemoglobin relative to saline-treatedmonkeys. In contrast, once weekly administration did not result indecreased hemoglobin levels relative to saline treatment.

Thus, in some embodiments, an IL-18 polypeptide (e.g., a wild typeIL-18, a stabilized IL-18 variant, a DR IL-18 variant, a D2D IL-18variant, a stabilized DR IL-18 variant, a stabilized D2D IL-18 variant,and the like) (e.g., SEQ ID NO: 19) is administered to an individual ata frequency of once per week (one dose per week) or less (e.g., once pertwo weeks or less, once per month or less). In some cases an IL-18polypeptide (e.g., a wild type IL-18, a stabilized IL-18 variant, a DRIL-18 variant, a D2D IL-18 variant, a stabilized DR IL-18 variant, astabilized D2D IL-18 variant, and the like) is administered to anindividual at a frequency in a range of from once per week to once per 6months (e.g., once per week to once per 4 months, once per week to onceper 2 months, or once per week to once per month). In some cases anIL-18 polypeptide (e.g., a wild type IL-18, a stabilized IL-18 variant,a DR IL-18 variant, a D2D IL-18 variant, a stabilized DR IL-18 variant,a stabilized D2D IL-18 variant, and the like) (e.g., SEQ ID NO: 19) isadministered to an individual at a frequency in a range of from once perweek to once per month.

In some cases, an IL-18 polypeptide (e.g., a wild type IL-18, astabilized IL-18 variant, a DR IL-18 variant, a D2D IL-18 variant, astabilized DR IL-18 variant, a stabilized D2D IL-18 variant, and thelike) (e.g., SEQ ID NO: 19) is administered to an individual about onceper week. In some cases, an IL-18 polypeptide (e.g., a wild type IL-18,a stabilized IL-18 variant, a DR IL-18 variant, a D2D IL-18 variant, astabilized DR IL-18 variant, a stabilized D2D IL-18 variant, and thelike) (e.g., SEQ ID NO: 19) is administered to an individual about onceevery two weeks. In some cases, an IL-18 polypeptide (e.g., a wild typeIL-18, a stabilized IL-18 variant, a DR IL-18 variant, a D2D IL-18variant, a stabilized DR IL-18 variant, a stabilized D2D IL-18 variant,and the like) (e.g., SEQ ID NO: 19) is administered to an individualabout once per month. In some cases an IL-18 polypeptide (e.g., a wildtype IL-18, a stabilized IL-18 variant, a DR IL-18 variant, a D2D IL-18variant, a stabilized DR IL-18 variant, a stabilized D2D IL-18 variant,and the like) is not administered to an individual more frequently thanonce per week.

Diseases

In some embodiments, the method of the present disclosure is useful fortreating or preventing a tumor or cancer tumors that have lost surfaceexpression of MHC class I; such as a tumor that has lost B2m, the MHClocus, or has mutations in other members of the antigen presentationand/or antigen loading complex, such as tapasin.

Metabolic diseases and disorders include various metabolic andendocrine-related diseases and disorders. The following are non-limitingexamples of metabolic and endocrine-related diseases and disorders thatcan be treated or prevented by the methods and compositions of theinvention: obesity, diabetes, prediabetes, type II diabetes, matureonset diabetes of the young (MODY), hyperglycemia, metabolic syndrome,dyslipidemia, hypertriglyceridemia, and hypercholesterolemia.

Non-limiting examples of other diseases and disorders that can betreated or prevented using the compositions and methods of the inventioninclude viral infections, bacterial infections, parasitic infections,and low immune activity. In some embodiments, the viral infection is atleast one of a pox viruses, a smallpox virus, molluscum contagiosum, HPVinfection, and warts caused by a virus. In some embodiments, theinfection is a systemic infection. In some embodiments, the viralinfection is a vaccinia virus infection. In some embodiments, the viralinfection is a systemic vaccinia virus infection. In some embodiments,the bacterial infection is sepsis. In some embodiments, the low immuneactivity is neutropenia, for example, as may occur with chemotherapy.

Non-limiting examples of other diseases and disorders that can betreated or prevented using the compositions and methods of the inventioninclude macular degeneration. For example, in some cases the disease ordisorder is wet macular degeneration, and in some cases the disease ordisorder is wet age-related macular degeneration. In some such cases,the IL-18 variant polypeptide (e.g., a stabilized IL-18) can be used asan anti-angiogenic. For example, a subject IL-18 variant polypeptide(e.g., a stabilized IL-18) can in some cases attenuate choroidalneovascularization.

In some embodiments, an IL-18 polypeptide (e.g., a stabilized IL-18 suchas a stabilized DR IL-18 variant or stabilized D2D IL-18 variant, andthe like) (e.g., SEQ ID NO: 19) are useful for the treatment orprevention of a disease or disorder. In various embodiments, the diseaseor disorder is cancer or a metabolic disease or disorder, includingobesity and diabetes (e.g., a subject method can cause a decrease inbody fat). Thus, in some embodiments, a composition comprises at leastone IL-18 polypeptide (e.g., a stabilized IL-18 such as a stabilized DRIL-18 variant or stabilized D2D IL-18 variant, and the like) (e.g., SEQID NO: 19). In other embodiments, a method of administering at least oneIL-18 polypeptide (e.g., a stabilized IL-18 such as a stabilized DRIL-18 variant or stabilized D2D IL-18 variant, and the like) (e.g., SEQID NO: 19), is performed to treat a disease or disorder, such as, butnot limited to, cancer or a metabolic disease or disorder.

The following are non-limiting examples of cancers that can be treatedor prevented by the methods and compositions of the disclosure: acutelymphoblastic leukemia, acute myeloid leukemia, adrenocorticalcarcinoma, appendix cancer, basal cell carcinoma, bile duct cancer,bladder cancer, bone cancer, brain and spinal cord tumors, brain stemglioma, brain tumor, breast cancer, bronchial tumors, burkitt lymphoma,carcinoid tumor, central nervous system atypical teratoid/rhabdoidtumor, central nervous system embryonal tumors, central nervous systemlymphoma, cerebellar astrocytoma, cerebral astrocytoma/malignant glioma,cerebral astrocytoma/malignant glioma, cervical cancer, childhood visualpathway tumor, chordoma, chronic lymphocytic leukemia, chronicmyelogenous leukemia, chronic myeloproliferative disorders, coloncancer, colorectal cancer, craniopharyngioma, cutaneous cancer,cutaneous t-cell lymphoma, endometrial cancer, ependymoblastoma,ependymoma, esophageal cancer, ewing family of tumors, extracranialcancer, extragonadal germ cell tumor, extrahepatic bile duct cancer,extrahepatic cancer, eye cancer, fungoides, gallbladder cancer, gastric(stomach) cancer, gastrointestinal cancer, gastrointestinal carcinoidtumor, gastrointestinal stromal tumor (gist), germ cell tumor,gestational cancer, gestational trophoblastic tumor, glioblastoma,glioma, hairy cell leukemia, head and neck cancer, hepatocellular(liver) cancer, histiocytosis, hodgkin lymphoma, hypopharyngeal cancer,hypothalamic and visual pathway glioma, hypothalamic tumor, intraocular(eye) cancer, intraocular melanoma, islet cell tumors, kaposi sarcoma,kidney (renal cell) cancer, langerhans cell cancer, langerhans cellhistiocytosis, laryngeal cancer, leukemia, lip and oral cavity cancer,liver cancer, lung cancer, lymphoma, macroglobulinemia, malignantfibrous histiocvtoma of bone and osteosarcoma, medulloblastoma,medulloepithelioma, melanoma, merkel cell carcinoma, mesothelioma,metastatic squamous neck cancer with occult primary, mouth cancer,multiple endocrine neoplasia syndrome, multiple myeloma, mycosis,myelodysplastic syndromes, myelodysplastic/myeloproliferative diseases,myelogenous leukemia, myeloid leukemia, myeloma, myeloproliferativedisorders, nasal cavity and paranasal sinus cancer, nasopharyngealcancer, neuroblastoma, non-hodgkin lymphoma, non-small cell lung cancer,oral cancer, oral cavity cancer, oropharyngeal cancer, osteosarcoma andmalignant fibrous histiocytoma, osteosarcoma and malignant fibroushistiocytoma of bone, ovarian, ovarian cancer, ovarian epithelialcancer, ovarian germ cell tumor, ovarian low malignant potential tumor,pancreatic cancer, papillomatosis, paraganglioma, parathyroid cancer,penile cancer, pharyngeal cancer, pheochromocytoma, pineal parenchymaltumors of intermediate differentiation, pineoblastoma and supratentorialprimitive neuroectodermal tumors, pituitary tumor, plasma cell neoplasm,plasma cell neoplasm/multiple myeloma, pleuropulmonary blastoma, primarycentral nervous system cancer, primary central nervous system lymphoma,prostate cancer, rectal cancer, renal cell (kidney) cancer, renal pelvisand ureter cancer, respiratory tract carcinoma involving the nut gene onchromosome 15, retinoblastoma, rhabdomyosarcoma, salivary gland cancer,sarcoma, sezary syndrome, skin cancer (melanoma), skin cancer(nonmelanoma), skin carcinoma, small cell lung cancer, small intestinecancer, soft tissue cancer, soft tissue sarcoma, squamous cellcarcinoma, squamous neck cancer, stomach (gastric) cancer,supratentorial primitive neuroectodermal tumors, supratentorialprimitive neuroectodermal tumors and pineoblastoma, T-cell lymphoma,testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroidcancer, transitional cell cancer, transitional cell cancer of the renalpelvis and ureter, trophoblastic tumor, urethral cancer, uterine cancer,uterine sarcoma, vaginal cancer, visual pathway and hypothalamic glioma,vulvar cancer, waldenstrom macroglobulinemia, and Wilms Tumor.

In some non-limiting examples, the cancer is defined not by thehistological tissue of origin, but by its molecular features. Forexample, tumors with high tumor mutational burden (TMB), tumors withmicrosatellite instability (MSI), tumors that lack or have diminishedsurface expression of MHC Class I (e.g., due to deletion of Beta-2microglobulin or Tapasin), or tumors that express high levels ofIL-18BP. In some cases, those molecular features are determined throughnext-generation sequencing (e.g., using TMB/MSI). In some cases, thosemolecular features are determined through immunohistochemistry,immunofluorescence, or flow cytometry. Thus, non-limiting examples ofcancers that can be treated or prevented by the methods and compositionsof the disclosure include solid tumor cancers, liquid cancers, bloodcancers, teratomas, sarcomas, and carcinomas.

In some embodiments, the compositions and methods of the disclosure areuseful for treating a tumor or cancer that is resistant to immunecheckpoint inhibitors (ICIs). Examples of immune checkpoint inhibitorsinclude, but are not limited to: anti-PD1 agents such as an anti-PD1antibody (e.g., nivolumab), anti-PD-L1 agents such as an anti-PD-L1antibody, anti-CTLA4 (e.g., ipilimumab), anti-TIM3, anti-TIGIT,anti-LAG3, anti-B7H3, anti-B7H4, anti-VISTA, anti-BTLA, anti-CD47,anti-SIRP alpha, anti-CD48, anti-CD155, anti-CD160, anti-TREM2,anti-IDO1, anti-Adenosine 2A receptor, anti-Aryl hydrocarbon receptor,anti-KIR, and anti-LILRB2. Examples of targets of immune checkpointinhibitors include but are not limited to: PD-L1, PD1, CTLA4, TIM3,TIGIT, LAG3, B7H3, B7H4, VISTA, BTLA, CD47, SIRP alpha, CD48, CD155,CD160, TREM2, IDO1, Adenosine 2A receptor, Aryl hydrocarbon receptor,KIR, and LILRB2.

Thus, examples of immune checkpoint inhibitors include agents thatinhibit proteins such as: PD-L1, PD1, CTLA4, TIM3, TIGIT, LAG3, B7H3,B7H4, VISTA, BTLA, CD47, SIRP alpha, CD48, CD155, CD160, TREM2, IDO1,Adenosine 2A receptor, Aryl hydrocarbon receptor, KIR, and LILRB2. Insome cases, a subject stabilized IL-18 variant polypeptide (e.g., aDR-IL-18 variant, a D2D-IL-18 variant) (e.g., SEQ ID NO: 19) isco-administered with an immune checkpoint inhibitor (e.g., an agent thatinhibits PD-L1, PD1, CTLA4, TIM3, TIGIT, LAGS, B7H3, B7H4, VISTA, BTLA,CD47, SIRP alpha, CD48, CD155, CD160, TREM2, IDO1, Adenosine 2Areceptor, Aryl hydrocarbon receptor, KIR, LILRB2, or any combinationthereof).

In some embodiments, the compositions and methods of the disclosure areuseful for treating a cancer that is associated with high levels ofIL-18BP (e.g., circulating or expressed by tumors) or a cancerassociated with a tumor in which IL-18R is expressed on infiltrating Tcells or NK cells.

Thus, the present invention relates to the prevention and treatment of adisease or disorder by administration of a therapeutically effectiveamount of an IL-18 variant polypeptide (e.g., a stabilized IL-18), arecombinant IL-18 variant polypeptide, an active IL-18 variantpolypeptide fragment (e.g., IL-18 variant peptide, etc.), to a cell,tissue, organ, or subject in need thereof, for the treatment of adisease or disorder, or its associated signs, symptoms or pathologies.In one embodiment, the prevent invention provides a method ofadministering a therapeutically effective amount of an IL-18 variantpolypeptide (e.g., a stabilized IL-18), a recombinant IL-18 variantpolypeptide, an active IL-18 variant polypeptide fragment (e.g., IL-18variant peptide, etc.), to a subject having, suspecting of having, or atrisk for having, a disease or disorder described herein. In oneembodiment, the prevent invention provides a method of administering atherapeutically effective amount of an IL-18 variant polypeptide (e.g.,a stabilized IL-18), a recombinant IL-18 variant polypeptide, an activeIL-18 variant polypeptide fragment (e.g., IL-18 variant peptide, etc.),to a cell, tissue or organ of a subject having, suspecting of having, orat risk for having, a disease or disorder described herein.

Kits

The present invention also pertains to kits useful in the methods of theinvention. Such kits comprise various combinations of components usefulin any of the methods described elsewhere herein, including for example,a IL-18 polypeptide (e.g., a stabilized IL-18 such as a stabilized DRIL-18 variant or stabilized D2D IL-18 variant, and the like) (e.g., SEQID NO: 19) and/or materials for quantitatively analyzing IL-18 variantpolypeptide or IL-18 variant nucleic acid, and/or materials forassessing the activity of an IL-18 variant polypeptide or an IL-18variant nucleic acid, and/or instructional material. For example, insome embodiments, the kit comprises components useful for thequantification of IL-18 variant nucleic acid in a biological sample. Inanother embodiment, the kit comprises components useful for thequantification of IL-18 variant polypeptide in a biological sample. In afurther embodiment, the kit comprises components useful for theassessment of the activity (e.g., enzymatic activity, ligand bindingactivity, etc.) of an IL-18 variant polypeptide in a biological sample.

In a further embodiment, the kit comprises the components of an assayfor monitoring the effectiveness of a treatment administered to asubject in need thereof, containing instructional material and thecomponents for determining whether the level of IL-18 signaling in abiological sample obtained from the subject is modulated during or afteradministration of the treatment. In various embodiments, to determinewhether the level of IL-18 signaling is modulated in a biological sampleobtained from the subject, the level of IL-18 signaling is compared withthe level of at least one comparator control contained in the kit, suchas a positive control, a negative control, a historical control, ahistorical norm, or the level of another reference molecule in thebiological sample. In certain embodiments, the ratio of IL-18 signalingand a reference molecule is determined to aid in the monitoring of thetreatment.

EXAMPLES

The invention is further described in detail by reference to thefollowing experimental examples. These examples are provided forpurposes of illustration only, and are not intended to be limitingunless otherwise specified. Thus, the invention should in no way beconstrued as being limited to the following examples, but rather shouldbe construed to encompass any and all variations which become evident asa result of the teaching provided herein.

Without further description, it is believed that one of ordinary skillin the art can, using the preceding description and the followingillustrative examples, make and utilize the present invention andpractice the claimed methods. The following working examples thereforeare not to be construed as limiting in any way the remainder of thedisclosure.

Example 1: IL-18 Variant Polypeptides

IL-18 is a pro-inflammatory cytokine that can stimulate T, NK, andmyeloid cells. It has been proposed as an immunotherapeutic agent forcancer given its ability to stimulate anti-tumor immune cells. Asdemonstrated herein, the therapeutic efficacy of recombinant IL-18treatment can be greatly limited by upregulation of its naturalendogenous soluble inhibitor IL-18BP. The present disclosure is based,in part, on the development of variants of both human and mouse IL-18that exhibit minimal binding to IL-18BP. The cytokine variants exhibitaltered relative preference for the receptors (IL-18Rα and IL-18BP) byhundreds of thousands to over a million-fold. These variants have potentanti-tumor activity in preclinical tumor models, both as monotherapiesand in combination with immune checkpoint inhibitors such as anti-PD-1.As an additional application, IL-18 also has a well-establishedanti-obesity role and it is demonstrated herein that administration ofthe variants greatly reduces body fat composition compared to WT IL-18treatment. The new variants thus have indications inendocrinology/metabolism/obesity in addition to tumor immunotherapies.

Also described herein are an additional set of IL-18 variants that actas IL-18BP antagonists by exclusively binding IL-18BP with absent orgreatly reduced binding to IL-18Ra. It is envisaged that these proteinscould be used to enhance the activity of endogenous IL-18 byneutralizing IL-18BP.

The materials and methods employed in these experiments are nowdescribed.

Protein Expression and Purification

Human IL-18, mouse IL-18 (amino acids 1-157) and variants thereof, wereassembled as gBlocks (Integrated DNA Technologies, IDT) and cloned intoa pET28a-smt vector for expression of N-terminal sumo-tagged andC-terminal hexahistidine-tagged proteins in E. coli BL21 (DE3) Rosettastrain. Protein expression was induced with 0.5 mM IPTG at 16° C. for 20hours. The fusion proteins were first purified using Ni-chelatingresins, followed by cleavage of the sumo tag with sumo protease.Proteins were then separated from aggregates by successive ammoniumsulfate cuts, with aggregates precipitating at 20% ammonium sulfate andthe target proteins at 70% ammonium sulfate. Protein pellets wereresuspended and applied to Ni-chelating resins again to remove sumotags, and were subjected to an endotoxin removal wash with 0.1% TritonX-114. Finally, eluted protein was buffer exchanged to PBS by PD-10column (GE Healthcare). Protein sample was tested for monodispersity bysize exclusion chromatography using an FPLC (Bio-Rad) and SEC650 column(Bio-Rad).

Human IL-18Rα ectodomain (amino acids 19-329), IL-18Rβ ectodomain (aminoacids 15-356), and IL-18BP (amino acids 31-194), were secreted andpurified via a baculovirus expression system. In brief, all constructsequences were cloned into the pAcBN-BH3 vector (BD Biosciences) with anN-terminal gp67 signal peptide and a C-terminal AviTag™ andhexahistidine tag. Spodoptera frugiperda (Sf9) insect cells cultured at27° C. in SF900 II SFM medium (Invitrogen) were transfected with theplasmid constructs to establish high-titer recombinant virus, which wassubsequently amplified. Trichopulsia ni (High-Five) insect cells(Invitrogen) grown in Insect Xpress medium (Lonza) at 27° C. wereinfected with the viruses to express recombinant protein. Three daysafter infection, proteins were extracted via Ni-NTA (QIAGEN) affinitychromatography, concentrated, and purified to >98% homogeneity withSEC650 sizing column (Bio-Rad) equilibrated in 10 mM HEPES (pH 7.5) and150 mM NaCl.

Mouse IL-18Rα ectodomain (amino acids 19-329) and IL-18BP (amino acids31-194) were produced as secreted proteins using the Expi293 expressionsystem (Thermo Fisher). In brief, all construct sequences were clonedinto the BacMam expression vector pEZT_D_Lux with an N-terminal H7signal peptide and a C-terminal AviTag™ and hexahistidine tag. Expi293cells cultured at 37° C. in Expi293 expression medium (Thermo Fisher)were transfected with plasmids using the ExpiFectamine 293 TransfectionKit (Thermo Fisher) according to the manufacturer's instructions. Cellswere harvested 3-5 days after transfection. Protein purificationprocedures were the same as with the human proteins.

For protein biotinylation, a C-terminal biotin acceptor peptide(AviTag)-GLNDIFEAQKIEWHE was fused to all IL-18 receptor constructs.Protein biotinylation was carried out with soluble BirA ligase enzyme in0.1 mM Bicine (pH 8.3), 10 mM ATP, 10 mM magnesium acetate, and 0.5 mMbiotin (Sigma). Proteins were purified by size exclusion on a SEC650column, as described above.

Yeast Display of IL-18

Human and mouse IL-18 gene block (IDT) were synthesized and cloned intothe vector pYAL and displayed on the Saccharomyces cerevisiae strainEBY100. Individual colonies of IL-18 yeast were grown overnight at 30°C. in SDCAA liquid media and induced in SGCAA liquid media for 1 day at20° C. IL-18 display levels on yeast were verified by flow cytometryusing an anti-cMyc tag antibody (anti-myc-PE; Cell SignalingTechnologies). Receptor staining with biotinylated IL-18Rα (with orwithout IL-18R β) or biotinylated IL-18BP was performed in PBSsupplemented with 0.5% BSA and 2 mM EDTA (PBE) on ice. All analysis wasperformed on a Sony SA3800 flow cytometer.

Human IL-18 Library Construction and Selection

For the first human decoy-resistant IL-18 library, fourteen hIL-18Rα andhIL-18BP contact residues in hIL-18 (Table 1) were identified fromhomologous positions by aligning the structure ofhIL-18/hIL-18Ra/hIL-18Rβ complex (Protein Data Bank (PDB ID) code 3OW4)to the structure of IL-18/IL-18BP (PDB ID 3F62). A library randomizingthese residues was constructed using assembly PCR with the degenerateprimers listed in Table 2. The library had a theoretical diversity of˜1.96×10¹¹ unique protein sequences. The PCR products were furtheramplified with primers having homology to the pYAL vector andco-electroporated together with linearized pYAL into EBY100 yeast. Theresulting library contained 2.5×10⁸ transformants. For the second V2.0human decoy-resistant IL-18 library, eleven hIL-18Rα and hIL-18BPcontact residues in hIL-18 were selected to randomize, with atheoretical diversity of 3.44×10⁹ variants (described in FIG. 7A). Alibrary randomizing these residues was constructed using assembly PCRwith the degenerate primers and co-electroporated with pYAL into EBY100yeast. The resulting library had a diversity of 6×10⁸ transformants.

Table 1 shows the First Human IL-18 library design

Residue Codon Potential residues 1Y YNT Y, F, S, C, L, P, H, R L5 NWT L,F, I, Y, H, N, V, D 8K MRA K, R, R, Q 51M RNS M, I, T, N, K, S, R, V, A,D, E, G 53S ARA K, R 55S RRW S, R, G, G, N, K, D, E 59G RNA G, E, A, V,I, T, K, R 60M VDG M, K, R, L, Q, R, V, E, G 103Q VAW Q, K, E, D, N, H105S RRW S, K, R, N, D, E, G, G 110D VAW D, E, K, N, Q, H 111N NAT N, D,H, Y 153V RHT V, A, D, I, T, N 155N VAW N, K, D, E, Q, H

Table 2 shows the First human IL-18 library assembly primers

Primer Sequence (5′ to 3′) hIL18Lib1CATTTTCATTAAGATGCAGTTACTTCGCTGTTTTTCAATATTTTCTGTTATTGCTAGC (SEQ ID NO: 194) hIL18Lib2AATTACGGATGACCGAAAGTYKGGATTCAWNCTTGCCGAAANRTGCTAAAACGCTAGCAATAACAGAAAATATT GAAAAA (SEQ ID NO: 195) hIL18Lib3ACTTTCGGTCATCCGTAATTTGAACGACCAAGTCCTT TTTATTGACCAGGG (SEQ ID NO: 196)hIL18Lib4 ACTATCCGTCATATCCTCGAATAAGGGACGATTGCCCTGGTCAATAAAAAGGACT (SEQ ID NO: 197) hIL18Lib5CTTATTCGAGGATATGACGGATAGTGATTGCCGTGAC AACGCCC (SEQ ID NO: 198) hIL18Lib6ACTGAGATTGTTACCGCCHBTNYACGGGGTTGWYYATCTYTATASNYAGAGATGATGAAAATTGTACGAGGGGC GTTGTCACGG (SEQ ID NO: 199)hIL18Lib7 GGCGGTAACAATCTCAGTTAAGTGCGAAAAAATCTCGACACTTTCTTGTGAA (SEQ ID NO: 200) hIL18Lib8GGTTCATTTCCTTGAACGAAATGATCTTGTTTTCACA AGAAAGTGTCGAGATT (SEQ ID NO: 201)hIL18Lib9 CATTTCGTTCAAGGAAATGAACCCGCCGGATAATATCAAGGATACAAAATCAGATATTATTT (SEQ ID NO: 202) hIL18Lib10TGATGAGCTCTCGAATTGCATCTTATNWTBGTGTCCAGGCACWYYACGWTBGAAGAAAATAATATCTGATTTTG TATCCTTGATATTA (SEQ ID NO: 203)hIL18Lib11 ATAAGATGCAATTCGAGAGCTCATCATACGAAGGTTACTTTTTAGCCTGCG (SEQ ID NO: 204) hIL18Lib12AATTAACTTAAACAGGTCGCGCTCCTTCTCGCAGGCT AAAAAGTAACCTT (SEQ ID NO: 205)hIL18Lib13 GCGACCTGTTTAAGTTAATTCTTAAGAAAGAAGATGAGTTGGGGGATCG (SEQ ID NO: 206) hIL18Lib14CCAGAACCACCGTCCTCWTBCTGADYGGTAAACATGA TGCTACGATCCCCCAACTCATCTT(SEQ ID NO: 207) hIL18Lib15 GAGGACGGTGGTTCTGGATCCGAACAAAAGCTTATCTCCGAAGAAGACTTGG (SEQ ID NO: 208) hIL18Lib16CCACCAGATCCACCACCACCCAAGTCTTCTTCGGAGA TA AG (SEQ ID NO: 209)

For both libraries, transformed yeast were recovered and expanded inliquid synthetic dextrose medium with casamino acids (SDCAA) medium at30° C. and induced by dilution 1:10 into liquid synthetic galactosemedium with casamino acids (SGCAA) medium and cultured at 20° C. for 24hours. Appropriate numbers of induced yeast were used in each round toensure at least 10-fold coverage of the expected diversity of thelibrary at each step, and not less than 10⁸ cells. All selection stepswere carried out at 4° C. using PBE buffer (PBS with 0.5% BSA and 2 mMEDTA). For the first generation library, each round's selection reagentsare listed in Table 3. For round 1, yeast were counter-selected withanti-Cy5/AlexaFluor 647 microbeads (Miltenyi) and an LS MACS column(Miltenyi) to remove non-specific bead binders. Positive selection wasperformed by labeling yeast with 1 μM biotinylated hIL-18Rα for 1 hourat 4° C., followed by magnetic selection with SA/AlexaFluor 647microbeads and an LS MACS column. For round 2, counter-selection wasperformed with 1 μM biotinylated IL-18BP, with positive selectionidentical to round 1. For rounds 3-5, selection was performed byincubating yeast with 100 nM (rounds 3-4) or 10 nM (round 5)biotinylated IL-18Rα and 250 nM pre-formed, biotin-capped hIL-18BP/SA-PEtetramers. After competition binding, yeast were washed and labeled withSA AlexaFluor 647 to detect IL-18Ra. Display levels were determined bystaining with AlexaFluor 488-conjugated anti-cMyc (Cell SignalingTechnologies), and the top 1% of display-normalized IL-18Rα binders (outof IL-18BP non-binders) were isolated using FACS with a Sony SA3800 cellsorter. After each round of selection, recovered yeast were expanded inSDCAA medium at 30° C. overnight and later induced at 20° C. by a 1:10dilution into SGCAA medium for 24 hours.

The V2.0 human DR-IL-18 library was selected in a similar fashion, withspecific selection steps elaborated in FIG. 7B.

Mouse IL-18 Library Construction and Selection

Construction and selection procedures are similar to human IL-18, withthe following changes. Library construction was informed by an in-silicomodeled mouse IL-18/receptor complex structure (predicted by Phyer2.0).Thirteen positions were chosen for randomization (Table 3) using primersdescribed in Table 4. Co-electroporation with pYAL yielded a library of4×10⁸ transformants. Selection reagents used for each round are listedin Table 5.

Table 3 shows the Mouse IL-18 library design

Residue Codon Potential residues 1N NWT F, Y, L, H, I, N, V, D 50M RNSM, I, T, N, K, S, R, V, A, D, E, G 51Y NRN Y, K, R, D, E 52K VNS L, P,H, Q, R, I, M, T, N, K, S, V, A, D, E, 54S RRW S, R, G, G, N, K, D, E55E VRN E, K, N, R, S, R, H 56V VNV V, S, P, T, A, K, R 57R RVW R, D, E,S, T 58G RNA G, E, A, V, I, T, K, R 59L VDR L, K, R, Q, R, V, E, G 104RNDH R, D, E, N, Y, F, I, L, V 109N NAT N, D, H, Y 151L VHY L, V, A, D,I, T, N

Table 4 shows the Mouse IL-18 library assembly primers

Primer Sequence (5′ to 3′) mIL18lib1CATTTTCATTAAGATGCAGTTACTTCGCTGTTTTTCAATATTTTCTGT TATTGCTAGCGTTT (SEQ ID NO: 210) mIL18lib2TTGTACAGTGAAGTCGGCCAAAAWNTGCTAAAACGCTAGCAATAAC AGAAAATAT (SEQ ID NO: 211) mIL18lib3GCCGACTTCACTGTACAACCGCAGTAATACGGAATATAAATGACCA AGTTCTCTTCGTT (SEQ ID NO: 212) mIL18lib4TTGATCAATATCAGTCATATCCTCGAACACAGGCTGTCTTTTGTCAA CGAAGAGAACTTGGTCATTT (SEQ ID NO: 213) mIL18lib5GTGTTCGAGGATATGACTGATATTGATCAAAGTGCCAGTGAACCCCA GACCAGA (SEQ ID NO: 214) mIL18lib6TCACAGAGAGGGTCACAGCYHBTNYWBYBNBNYBWYYGTCSNBNY NSNYGTATATTATCAGTCTGGTCTGGGGTTCAC (SEQ ID NO. 215) mIL18lib7GCTGTGACCCTCTCTGTGAAGGATAGTAAAATGTCTACCCTCTCCTG TAAGAACAAGA (SEQ ID NO: 216) mIL18lib8GTATATCATCAATATTTTCAGGTGGATCCATTTCCTCAAAGGAAATG ATCTTGTTCTTACAGGAGAGGG (SEQ ID NO: 217) mIL18lib9AATGGATCCACCTGAAAATATTGATGATATACAAAGTGATCT CATATTCTTTCAGAAANDHGTTCCAGGACACNATAAGATGGAGTTTG mIL18lib9AATGGATCCACCTGAAAATATTGATGATATACAAAGTGATCTCATATTCTTTCAGAAANDHGTTCCAGGACACNATAAGATGGAGTTTGAATCT TCACT (SEQ ID NO: 218) mIL18lib10CCTTTTGGCAAGCAAGAAAGTGTCCTTCATACAGTGAAGATTCAAAC TCCATCTTAT (SEQ ID NO: 219) mIL18lib11CTTTCTTGCTTGCCAAAAGGAAGATGATGCTTTCAAACTCATTCTGA AAAAAAAGGATGA (SEQ ID NO: 220) mIL18lib12CCACCACTTTGATGTAAGTTAGTRDBAGTGAACATTACAGATTTATC CCCATTTTCATCCTTTTTTTTCAGAATGAG (SEQ ID NO: 221) mIL18lib13ACTAACTTACATCAAAGTGGTGGTTCTGGATCCGAACAAAAGCTTAT CTCCGAAGAAGA (SEQ ID NO: 222)

Table 5 shows the summary of library selection reagents

Human IL-18 library selection Mouse IL-18 library selectionCounter-selection Positive Selection Counter-selection PositiveSelection Round1 SA-beads alone 1 μM hIL-18Rα- — 1000 nM IL-18Rα-SA-beads SA-beads Round2 1 μM IL-18BP 1 μM IL-18Rα- —  1 μM IL-18RαSA-beads Round3 1 μM IL-18BP 100 nM hIL-18Rα 1 μM IL-18BP  1 μM IL-18RαRound4 1 μM IL-18BP  10 nM hIL-18Rα 1 μM IL-18BP 100 nM IL-18Rα  Round5250 nM IL-18BP  10 nM hIL-18Rα 1 μM IL-18BP 10 nM IL-18Rα tetramerRound6 — — 250 nM IL-18BP 200 nM IL-18Rα  tetramerSurface Plasmon Resonance

Experiments were conducted using a Biacore T100 and carried out at 25°C. Biotinylated IL-18Rα or IL-18BP were immobilized onto a Biacorebiotin capture chip (Series S CAP sensor chip, GE Healthcare) to yieldan Rmax of ˜50 RU (IL-18Rα) or ˜10 RU (IL-18BP). Measurements were madewith serial dilutions of the IL-18 variants in HEPES buffered Saline-P+buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 0.005% surfactant P20). Thesurface was regenerated by three 60-sec injections of regenerationbuffer (3/4 (v/v) 8M guanidine hydrochloride with 1/4 (v/v) 1M sodiumhydroxide). Experiments were performed in multiple channelssimultaneously for increased observations. All data were analyzed withthe Biacore T100 evaluation software version 2.0 with a 1:1 Langmuirbinding model.

Cell Lines

HEK-Blue IL-18 sensor cells (InvivoGen) were maintained in completemedia (DMEM containing 10% heat-inactivated FBS, 2 mM L-glutamine, 50U/mL penicillin, and 50 μg/mL streptomycin) supplemented with 100 μg/mLNormocin, 30 μg/mL Blasticidin, 180 μg/mL Zeocin, and 200 μg/mLHygromycin. YUMMER1.7 melanoma cells were cultured and prepared aspreviously described (Wang et al., 2017, Pigment Cell Melanoma Res.,30(4):428-435).

HEK-Blue Cytokine Activity Assay

For cytokine activity measurements, 50,000 HEK-Blue IL-18 sensor cellsper well of a flat-bottom 96-well plate were incubated with recombinanthuman IL-18 at successively decreasing concentrations in a total volumeof 200 μL of complete media. After 20-24 hours of incubation at 37° C.and 5% CO₂, 30 μL of cell culture supernatant was mixed with 170 μLQUANTI-Blue detection media (InvivoGen) and incubated at 37° C. and 5%CO2 until a color change from pink to blue was detectable (0.5-4 hours).Levels of alkaline phosphatase were quantified using a spectrophotometerat 655 nm wavelength. Cytokine activity was determined by calculatingthe relative absorbance value (percentage of the maximal absorbancevalue measured at 655 nm) for each cytokine in the assay.

For IL-18BP blockade experiments, a fixed concentration of recombinanthuman IL-18 was pre-incubated with recombinant human IL-18BP atsuccessively decreasing concentrations for 1 hour at 4° C. Subsequently,the protein mixture was added to the HEK-Blue IL-18 sensor cells and theassay was performed as described.

Mice

C57BL/6 wild type mice (6-9 weeks old) from Jackson Laboratory were usedfor in vivo mouse experiments. Experimental groups were matched byweight, sex, and age. All animal experiments were conducted incompliance with approval from the Yale Institutional Animal Care and UseCommittee.

In Vivo Pharmacodynamic and Pharmacokinetic Studies

Mice (n=9 per group) received daily intraperitoneal (i.p.) injections of1 mg/kg recombinant IL-18 (WT or variant SEQ ID NO: 61), or PBS asvehicle control. On day 1, day 4, and day 7 of the experiment, 3 miceper group were sacrificed 5 hours post-injection for blood collectionvia cardiac puncture, and subsequent analysis of blood plasma or whiteblood cells (see mouse IL-18BP ELISA, Luminex-based multipleximmunoassay for mouse cytokine analysis, as well as immunophenotypingvia flow cytometry) was performed. Throughout the 7 days of theexperiment, body temperatures were monitored daily using the Rodentthermometer BIO-TK8851 (Bioseb) and the RET 3 rectal probe for mice(Braintree Scientific Inc.). Body weights were monitored daily.

Plasma Preparation from Whole Blood

Plasma preparation from whole blood was performed using EDTA-coatedMicrotainer Plasma Separator Tubes (BD) according to manufacturer'sinstruction. Plasma samples were frozen once at −20° C. before beingused for analytical assays.

IFN-γ and IL-18BP ELISA

To measure levels of human IFN-γ in cell culture supernatant, the HumanIFN-γ ELISA MAX Deluxe Set (BioLegend) with a sensitivity of 4 pg/mL anda detection range of 7.8-500 pg/mL was used according to themanufacturer's instructions. For quantification of human IL-18BP in cellculture supernatant, the Quantikine Human IL-18BP Immunoassay (R&DSystems) with a sensitivity of 7.52 pg/mL and a detection range of26.6-1,700 pg/mL was used. Mouse IL-18BP levels in blood plasma werequantified using the Mouse IL-18BP ELISA Kit (R&D systems) with asensitivity of 0.156 ng/mL and a detection range of 0.156-10 ng/mL. Allassays including sample preparation were performed according tomanufacturer's instructions.

Luminex-Based Multiplex Immunoassay for Mouse Cytokine Analysis

To quantify a variety of mouse cytokine levels in blood plasma includingIFN-γ and IL-12, the luminex-based Bio-Plex Pro multiplex immunoassay(Bio-Rad) was performed using the Bio-Plex 200 System (Bio-Rad).Cytokines of interest were analyzed using the Bio-Plex Pro MouseCytokine Standard 23-Plex (Group I) reconstituted in DMEM, following themanufacturer's instructions.

Immunophenotyping Via Flow Cytometry

For white blood cell analysis, 100 μL of whole blood were collected intoan EDTA-coated Microtainer Plasma Separator Tube (BD) additionallycontaining 50 μL Heparin-solution, and mixed by inverting several times.Red blood cell lysis was performed by adding ACK Lysing Buffer (VWR) andincubating for 3-5 minutes at room temperature. After adding MACS buffer(2 mM EDTA, 2% FBS, in PBS), white blood cells were collected bycentrifugation (5 minutes, 400×g, 4° C.) and aspiration of thesupernatant. White blood cells were washed once with cold MACS buffer,and collected again as described. The cell pellet was resuspended in 200μL MACS buffer containing 10% (v/v) rat serum (STEMCELL TechnologiesInc.) and specific fluorescently-labeled antibodies to stain forsubsequent flow cytometric analysis. Staining was performed for 30minutes at 4° C. using the following antibodies: αCD4-AF700 (BioLegend),αCD8-APC (BioLegend), B220-APC-Cy7 (BioLegend), CD11b-PB (BioLegend),NK1.1-PE (BioLegend), NKp46-PE (BioLegend), and CD69-FITC (BioLegend).Thereafter, white blood cells were washed twice with MACS buffer asdescribed before. Finally, the cells were resuspended in 100 μL MACSbuffer and samples were acquired using the flow cytometer (Sony SA3800).An aliquot of 10 μL was taken to perform cell counting using theInvitrogen Countess II Automated Cell Counter (Thermo FisherScientific). FlowJo v10.3 software was used for data analysis, and cellswere gated for leukocytes and single events using the forward and sidescatter.

Tumor Treatment Experiments

0.5×10⁶ YUMMER1.7 cells were implanted subcutaneously into C57BL/6Jmice. 7 days after implantation, when tumors were approximately 50 mg,treatment was initiated. Mice were divided into treatment cohorts whichincluded: 1) vehicle (saline), 2) anti-PD1 (rat clone RMP1-14, Bio XCell, West Lebanon, New Hampshire, US), 3) wildtype IL-18, 4) SEQ ID NO:61, 5) wild type IL-18+anti-PD-1, and 6) SEQ ID NO: 61 IL-18+anti-PD1.Anti PD-1, wild type IL-18, and SEQ ID NO: 61 IL-18 were administeredvia intraperitoneal injection twice weekly at 8 mg/kg, 0.32 mg/kg, and0.32 mg/kg, respectively. Mice were monitored for signs of clinicaltoxicity, and tumor growth was tracked twice weekly using calipermeasurements. Mice were euthanized when the tumor diameter reached orexceeded 1.5 cm in greatest dimension; this was considered the endpointfor survival analyses.

B2m-deficient YUMMER1.7 studies were conducted in a similar fashion,with the minor changes. 1.0×10⁶ cells were engrafted, as tumors grewslower than the parental strain. Treatments consisted of saline,anti-PD1 plus anti-CTLA4, and SEQ ID NO: 61 given at the same scheduleand dose as the studies above.

The results of the experiments are now described.

The IL-18 Axis as a Target for Cancer Immunotherapy

To identify potential signaling nodes for immunotherapeuticintervention, single cell RNAseq data from tumor infiltratinglymphocytes was analyzed for the expression of cytokine pathwaycomponents (Singer et al., 2016, Cell, 166:1500-1511, e1509). Thereceptor subunits for IL-18-IL-18Rα (i.e., IL-18R1) and IL-18Rβ (i.e.,IL-18RAP)—as well as IL-18 itself were upregulated in both activated anddysfunctional lymphocyte programs (FIG. 1A).

Further analysis of the Immunological Genome (ImmGen) database revealedthat expression of both IL-18 receptor subunits correlated withexpression of T cell “exhaustion” markers in CD4 and CD8 cells includingPD-1, Tim3, Lag3, and TIGIT following chronic antigen exposure as shownin FIG. 1B. These expression features suggested that the IL-18 pathwaycould be used to selectively stimulate activated anddysfunctional/exhausted T cells within tumors as an immunotherapeuticparadigm. IL-18 is a Th1 cytokine initially termed“interferon-gamma-inducing-factor” (IGIF) for its ability to robustlystimulate release of interferon gamma (IFN-γ) by T and NK cells.Feedback inhibition of IL-18 is achieved by IFN-γ-driven induction ofIL-18BP, a high-affinity secreted decoy receptor for IL-18 thatsterically hinders IL-18's ability to bind and activate its receptor(FIG. 2A). Without wishing to be bound by any particular theory, thismechanism is reminiscent of the induction of PD-L1 by IFN-γ, suggestingthat IL-18BP may act as a “soluble immune checkpoint.” Consistent withthis hypothesis, it was found that IL-18BP is unregulated in severaltypes of cancer, most notably breast, gastric, and brain cancer in theTCGA and Oncomine databases (FIG. 2B). Furthermore, IL-18BP expressionstrongly correlates with expression of the crucial immune checkpointPD-1 in tumors (r=0.65 and 0.78 in gastric and breast cancerrespectively, FIG. 2C), suggesting that IL-18BP may also contribute totumor immune evasion and lymphocyte exhaustion.

Recombinant IL-18 has been administered to cancer patients in multipleclinical trials. It was found to be well-tolerated even at high doses of2 mg/kg, with robust pharmacodynamics outputs including expansion ofactivated CD69⁺ natural killer (NK) cells and dramatic increases inserum IFN-γ levels. However, a phase II trial of melanoma patients wasdiscontinued due to lack of efficacy. Examination of the reportedpharmacodynamics results from these clinical trials reveals that theeffectiveness of rIL-18 wanes with repeated dosing, with tachyphylaxisseen with respect to peripheral NK cell activation/expansion andcytokine release (including IFN-γ and GM-CSF). The waning effectivenessof rIL-18 coincides with a profound increase in the serum levels ofIL-18BP, more than two orders of magnitude over pre-treatment levels andoften exceeding 100 ng/mL. Without wishing to be bound by any particulartheory, it was hypothesized that IL-18BP limits the effectiveness ofrIL-18 therapy and that IL-18 variants that are impervious to IL-18BPinhibition could be effective tumor immunotherapies. Additionally,inhibitors of IL-18BP will likely be effective for tumor immunotherapy.

Engineering IL-18 Variants that are Resistant to IL-18BP Inhibition(Human DR-IL-18 Variants)

To obtain variants of IL-18 that can signal through IL-18Rα/IL-18Rf3,but are impervious to inhibition by IL-18BP, directed evolution withyeast surface display was utilized. The structure of the ternarysignaling complex of human IL-18:IL-18Rα:IL-18Rf3 (PDB=30W4) was firstanalyzed, and residues of IL-18 that have a shared interface with thesignaling complex and IL-18BP were identified (FIG. 3A). As thestructure of hIL-18:hIL-18BP has not been determined, a related complexbetween IL-18 and a viral (ectromelia virus) orthologue of IL-18BP wasutilized (PDB=3F62). A combinatorial library randomizing this set ofresidues to a defined set of alternatives (see Table 1) was createdusing degenerate oligonucleotide primers and assembly PCR. This librarywas electroporated into yeast together with the N-terminal yeast displayvector pYAL to obtain a library with 2.5×10⁸ transformants. Using thislibrary, directed evolution was performed by conducting successiverounds of selection using magnetic and fluorescent cell sorting (FACS)with recombinant hIL-18Rα and counterselection with hIL-18BP, assummarized in FIG. 3B. After five rounds of selection, the clearmajority of the library clones had completely swapped their relativepreference for hIL-18BP and hIL-18Rα as compared to WT hIL-18 (FIG. 3C).These clones were designated as “DR-hIL-18” variants, where “DR” standsfor “decoy-resistant.”

Sequencing of 96 clones from the post-round five pool revealed 21 uniquesequences, which were analyzed to create four “consensus sequences”, SEQID NO: 34-37 (FIG. 4 ). To estimate the binding affinities of thesevariants for hIL-18Rα and hIL-18BP, binding isotherms were establishedfor hIL-18Rα and IL-18BP binding using yeast-displayed cytokine variantsand flow cytometry. As seen in FIG. 5A, the DR-hIL-18 variants boundhIL-18Rα with comparable affinity to WT IL-18, but showed severelyattenuated binding to hIL-18BP, with apparent binding EC50 valuessignificantly greater than 1 μM. To additionally characterize thereceptor binding activities of the DR-IL-18 variants, the cytokines wereexpressed recombinantly and surface plasmon resonance for IL-18Rα andIL-18BP was performed (see FIG. 5B for representative traces). Theseresults are summarized in Tables 6 and 7 and demonstrate that theDR-hIL-18 variants have a dramatically decreased preference for IL-18BPcompared to IL-18Rα, by several orders of magnitude.

Table 6 depicket IL-18Rα and IL-18BP binding affinites of human IL-18variants by on-yeast binding isotherms.

Dissociation KD IL- KDratio: Constant Ratio IL-18 18Rα KD IL-IL-18BP/IL- normalized to Variant (M) 18BP (M) 18Rα WT IL-18 hIL-18 WT2.40E− 7.08E−09   2.95E−01 1 SEQ ID NO: 39 5.77E−NBD >3.47E+02 >1.17E+03 SEQ ID NO: 52 8.38E− NBD >2.39E+02 >8.09E+02 SEQID NO: 57 1.27E− NBD >1.57E+02 >5.34E+02 SEQ ID NO: 34 6.44E− 1.93E−05  3.00E+02   1.02E+03 SEQ ID NO: 35 9.15E− NBD >2.19E+02 >7.41E+02 SEQID NO: 36 1.13E− 1.16E−05   1.03E+02   3.48E+02 SEQ ID NO: 37 1.60E−NBD >1.25E+02 >4.24E+02 SEQ ID NO: 87  4.1E− NBD    4.9E+02  >7.2E+03SEQ ID NO: 88 N.D. 3.4E−07 — — SEQ ID NO: 89  1.7E− NBD    1.2E+03 >1.7E+04 SEQ ID NO: 90  4.2E− NBD    4.8E+02  >7.0E+03 SEQ ID NO: 91 3.7E− NBD    5.4E+02  >8.0E+03 NBD, no binding detected (20 uM used forratio calculations), — value not determined

Dissociation KD IL- KDratio: Constant Ratio IL-18 18Rα KD IL- IL-18BP/normalized to Variant (M) 18BP (M) IL-18Rα WT IL-18 hIL-18 WT 2.93E−091.90E−12 6.48E−04 1 SEQ ID NO: 39 — — — — SEQ ID NO: 52 — — — — SEQ IDNO: 57 — — — — SEQ ID NO: 34 8.05E−09 1.94E−08 2.41E+00 3.72E+03 SEQ IDNO: 35 1.31E−08 SEQ ID NO: 36 8.18E−09 1.86E−08 2.27E+00 3.50E+03 SEQ IDNO: 37 4.38E−09 1.83E−07 4.18E+01 6.45E+04 — indicates value notdetermined

Table 7 depicts IL-18Rα and IL-18BP binding affinities of human IL-18variants by SPR

Functional Characterization of Human DR-IL-18 Variants

A previous report from Kim et al (Kim et al., 2001, Proc Natl Acad SciUSA 98(6):3304-9) described 3 hIL-18 variants with enhanced activity andpurportedly decreased inhibition by IL-18BP: E42A, K89A, and E42A/K89A.These cytokine variants were displayed on yeast and IL-18BP inhibitionof IL-18Rα binding was assessed by flow cytometry. As seen in FIG. 6A,while the DR-hIL-18 variants were impervious to inhibition of hIL-18Rαbinding by hIL-18BP, the Kim et al variants showed roughly equivalenthIL-18BP neutralization as compared to WT hIL-18. These results indicatethat the DR-hIL-18 variants are IL-18BP independent, whereas the Kim etal variants are highly sensitive to IL-18BP inhibition, similar to WThIL-18.

To confirm that the DR-hIL-18 could yield productive signaling throughthe IL-18 receptor in a cellular context, concentration-responseexperiments were performed using the HEK-blue IL-18 reporter cell line.In this system, IL-18R signaling is read-out by expression of secretedalkaline phosphatase (SEAP) downstream of a Nfκb/AP1 promotor. In theabsence of IL-18BP, DR-hIL-18 variants yielded signaling EC50 valuescommensurate with WT hIL-18. However, the DR-hIL-18 variantsdemonstrated virtually no inhibition by hIL-18BP, with no detectableinhibition at 1 μM IL-18BP (FIG. 6B). Taken together, these studiesestablish that the DR-hIL-18 variants are biologically active andimpervious to IL-18BP neutralization in a cell signaling context.

Engineering and Characterization of Second-Generation Human IL-18Variants that are Resistant to IL-18BP Inhibition (Human v2.0 DR-IL-18Variants)

To obtain additional, potentially enhanced human DR-IL-18 variants, asecond library of human IL-18 randomized at 11 positions (FIG. 7A) wasdesigned and yeast was transformed as described above. The resultinglibrary of 6×10⁸ transformants was selected as outlined in FIG. 7B,yielding a robust preference for IL-18Rα compared to IL-18BP withsuccessive selection steps (FIG. 7C). 17 unique sequences were recoveredafter 5-6 rounds of selection (FIG. 8 ). As shown in FIG. 9A, whencompared to WT IL-18, clones SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO:91, and SEQ ID NO: 87 had equal or somewhat stronger binding to IL18Rαas measured by yeast-binding isotherms with biotinylated IL18Ra. Howeveras shown in FIG. 9B, these clones did not show any appreciable bindingto IL-18BP. FIG. 9C depicts the measurement of thermal stability byapplying a range of temperatures to the yeast-displayed clones showedthat they were more thermal stable than WT IL-18 by 7-13° C. Theseresults are summarized in FIG. 9D.

Engineering IL-18 Variants that are Resistant to IL-18BP Inhibition(Murine DR-IL-18 Variants)

As the human and mouse interspecies cross-reactivity of IL-18 forIL-18Rα is poor, murine equivalents of the DR-IL-18 variants that couldbe used for studies in mice were created. Similar to the approach takenfor hIL-18 above, a combinatorial library of mIL-18 variants randomizinga similar set of mIL-18Rα/mIL-18BP contact residues (Table 3) wascreated, yielding a library of 4×10⁸ transformants. Directed evolutionwas performed on this library similar to how it was performed with thehuman IL-18 library; the selection strategy is summarized in FIG. 10A.After the completion of six rounds of selection, the remaining cloneshad a near-complete preference for mIL-18Rα over mIL-18BP (FIG. 10B).Analysis of 96 clones revealed 11 unique sequences, from which werederived two consensus sequences SEQ ID NO: 60 and SEQ ID NO: 61 (FIG.10C). Yeast binding isotherms and surface plasmon resonance experimentsconfirmed these DR-IL-18 clones had an even greater independence forIL-18BP than the human IL-18 variants described herein, with themIL-18BP binding KD's being well above 1 μM, with mIL-18Rα bindingremaining roughly equal to WT mIL-18 (FIG. 11A, FIG. 11B, Tables 8 and9).

Table 8 depicts IL-18Rα and IL-18BP binding affinities of mouse IL-18variants by on-yeast binding isotherms

Dissociation KD IL- KD IL- KD ratio: Constant Ratio IL-18 18Rα 18BPIL-18BP/ normalized to Variant (M) (M) IL-18Rα WT IL-18 mIL-18 WT1.13E−08 2.13E−09   1.88E−01 1 SEQ ID NO: 70 1.35E−08NBD >7.41E+02 >3.93E+03 SEQ ID NO: 67 1.79E−08 NBD >5.59E+02 >2.96E+03SEQ ID NO: 64 4.20E−08 NBD >2.38E+02 >1.26E+03 SEQ ID NO: 62 4.30E−08NBD >2.33E+02 >1.23E+03 SEQ ID NO: 60 1.07E−08 NBD >9.35E+02 >4.96E+03SEQ ID NO: 61 1.13E−08 NBD >8.85E+02 >4.69E+03 NBD, no binding detected(10 μM used for ratio calculations)

Table 9 depicts IL-18Rα and IL-18BP binding affinities of mouse IL-18variants by SPR

Dissociation KD IL- KD IL- KD ratio: Constant Ratio IL-18 18Rα 18BPIL-18BP/ normalized to Variant (M) (M) IL-18Rα WT IL-18 mIL-18 WT6.00E−10 1.10E−12 1.83E−03 1 SEQ ID NO:70 2.20E−10 1.39E−05 6.32E+043.45E+07 SEQ ID NO: 67 7.00E−10 1.47E−05 2.10E+04 1.15E+07 SEQ ID NO: 641.69E−09 NBD >1.78E+04   >9.68E+06   SEQ ID NO: 62 1.09E−09 2.87E−052.63E+04 1.44E+07 SEQ ID NO: 60 5.40E−10 3.80E−06 7.04E+03 3.84E+06 SEQID NO: 61 7.90E−11 1.05E−05 1.33E+05 7.25E+07 NBD, no binding detected(30 μM used for ratio calculations)In Vivo Pharmacodynamic Studies of DR-IL-18 Variants

To assess the biologic effects of administration of the DR-IL-18variants in vivo, pharmacodynamics studies were performed in mice,comparing WT mIL-18 to SEQ ID NO: 61. In the first study, mice weretreated with vehicle (PBS), mIL-18 (1 mg/kg/day), or SEQ ID NO: 61 (1mg/kg/day) for a total of seven injections (FIG. 12A). Analysis ofperipheral blood phenotypes by flow cytometry showed that both WT mIL-18and SEQ ID NO: 61 increased peripheral NK cell numbers by over ten-fold,and peripheral monocyte counts by over five-fold compared to vehicletreatment; total CD4 and CD8 cell counts were not significantly affected(FIG. 12B). Examination of cellular activation status by CD69 inductionrevealed that SEQ ID NO: 61 treatment dramatically increased CD69 levelson CD4 and CD8 cells compared to mIL-18 or vehicle treatment; reachingover 30% and over 50% positivity for CD4 and CD8 subsets, respectively(FIG. 12C). While both mIL-18 and SEQ ID NO: 61 stimulated CD69expression on peripheral NK cells to over 20% positive by day 3, theCD69 levels decreased to non-significant levels for mIL-18 by day 6, butremained significantly elevated with SEQ ID NO: 61 treatment (FIG. 12C).Peripheral cytokine levels were also measured with a multiplexed Luminexpanel. As seen in FIG. 12D, both mIL-18 and SEQ ID NO: 61 increasedserum IFN-g, MIP1b, and G-CSF compared to vehicle treatment, but SEQ IDNO: 61 achieved much higher levels than mIL-18 by day 6 for each ofthese cytokines, as mIL-18 exhibited tachyphylaxis with plateaued ordecreasing induced cytokine levels with subsequent administration.

Effect of SEQ ID NO: 61 on Body Fat Composition

To assess the effect of the DR-IL-18 variants on body fat composition,we administered WT IL-18 at 1 mg/kg or 0.01, 0.1, or 1 mg/kg SEQ ID NO:61 by intraperitoneal injection to C57BL/6 mice every three days. Bodyfat and lean mass composition were monitored by echoMRI. All testeddoses of SEQ ID NO: 61 (1 mg/kg, 0.1 mg/kg, and 0.01 mg/kg) resulted instriking decreases in the overall percentage of body fat by day 12,while vehicle and mIL-18 treated mice did not have a significant changein total body fat composition (FIG. 13 , top). Specifically, SEQ ID NO:61-treated mice had either reduced or stable levels of total fat massduring the experiment (FIG. 13 , bottom left), but substantiallyincreased their total lean mass (FIG. 13 , bottom right). These resultsindicate that SEQ ID NO: 61, and other variants disclosed herein, couldbe used to therapeutically decrease body fat composition (e.g., fortreatment of obesity, diabetes, and/or metabolic syndrome).

Anti-Tumor Efficacy of DR-IL-18 Variants

The anti-tumor efficacy of DR-IL-18 (SEQ ID NO: 61) was assessed usingthe transplantable, syngeneic YUMMER1.7 malignant melanoma tumor model.WT mIL-18 and SEQ ID NO: 61 were administered intraperitoneally to micebearing YUMMER1.7 tumors biweekly at a dose of 0.32 mg/kg, with orwithout co-administration of anti-PD1 antibodies (8 mg/kg/q3d).Consistent with previous reports on its use in mice and humans, WT IL-18did not affect tumor growth or survival compared to vehicle (saline),and only marginally improved the efficacy anti-PD1 when administered incombination. However, SEQ ID NO: 61 cured 27% of treated mice as amonotherapy and produced a partial response in another 27%, an effectcommensurate with anti-PD1 treatment. The combination of SEQ ID NO: 61with anti-PD1 cured 80% of treated mice (FIG. 14A and FIG. 14B).

To establish the mechanism of action of DR-IL-18 on YUMMER1.7 tumors,cell depletion studies were performed using antibodies against CD8, CD4,NK1.1, and Interferon-gamma. As seen in FIG. 15A and FIG. 15B, depletionof CD8 cells or neutralization of Interferon-gamma completely abrogatedthe effectiveness of DR-IL-18. Depletion of CD4 cells did not affect theinitial activity of DR-IL-18 in terms of tumor growth, however, in CD4treated mice, therapeutic responses are not sustained, suggesting a roleof CD4 cells in supporting and sustaining anti-tumor immunity. Depletionof NK cells did not affect tumor growth or survival in YUMMER1.7 cells.

The activity of DR-IL-18 was additionally assessed in the immunogenicMC38 colorectal tumor model. A dose-finding study was first performed,administering saline, WT IL-18 (1 mg/kg twice weekly), or a range ofDR-IL-18 doses from 0.01 mg/kg, 0.1 mg/kg, or 1 mg/kg twice weekly. Asseen in FIG. 16 , WT IL-18 had no effect on tumor growth, whereasDR-IL-18 (SEQ ID NO: 61) showed dose-dependent efficacy, slowing tumorgrowth at 0.1 mg/kg and producing tumor regression at 1 mg/kg. Thecohorts were then expanded and potential synergism with immunecheckpoint inhibition was assessed. Again, WT IL-18 had no effect as amonotherapy and showed no enhancement of anti-PD1 efficacy. By contrast,DR-IL-18 showed robust monotherapeutic activity commensurate with orsuperior to anti-PD1, and the two therapies given together showedexceptional synergism, producing complete regression in all treated miceas seen in FIG. 17 .

To further characterize the mechanism of DR-IL-18, flow cytometricstudies were performed on the immune infiltrate of MC38 tumors from micetreated with saline, WT IL-18, or DR-IL-18 (SEQ ID NO: 61). Relative tosaline or WT IL-18, DR-IL-18 treatment increased CD8 and NK cellinfiltration per mg of tumor and additionally resulting in upregulationof activation markers of effector cells such as granzyme B and KLRG1(FIG. 18A, top row). Unlike other cytokine therapies such as IL-2 orIL-15, DR-IL-18 does not increase the CD8:Treg ratio within tumorscompared to saline treatment. However, DR-IL-18 treatment leads to amore favorable tumor immune microenvironment, by increasing the ratio ofCD8 cells to tumor associated macrophages (TAMs), and monocytic andgranulocytic myeloids derived suppressor cells (MDSCs). The secondarycytokine release profile was also measured from serum of the same miceusing a Luminex assay. As seen in FIG. 18B, DR-IL-18 treatment increasedsystemic levels of Interferon-gamma, IL-7, and IL-15 by over 100-foldrelative to WT IL-18 treatment. Taken in aggregate, these resultsindicate that DR-IL-18 produces anti-tumor efficacy through a uniquemechanism of action distinct from IL-2, IL-15, or WT IL-18 treatment.

Some of the secondary cytokines induced by DR-IL-18 therapy would bepredicted to potentially contribute to toxicity and/or decreasedeffectiveness. For instance, IL-17 which is upregulated>100-fold byDR-IL-18 contributes to colitis and psoriasis and additionallystimulates granulocytes that can become immunosuppressive myeloidderived suppressor cells. IL-5 and IL-13 are type 2 cytokines alsoupregulated by DR-IL-18 and could contribute to allergy, exacerbation ofasthma, or analphylaxis. Th2 T cells do not contribute toimmunotherapeutics responses and may promote immunosuppressive Tregdevelopment. As such, in certain instances the effectiveness and safetyof DR-IL-18 could be enhanced by selective inhibition of undesiredsecondary cytokines such as IL-17, IL-5, and IL-13, for instance by aneutralizing antibody.

Many tumors are resistant to immune checkpoint inhibition, either atinitial presentation (primary resistance) or after an initial responseto treatment (secondary resistance). The most prevalent cause ofresistance of checkpoint inhibitors is loss of antigen presentationthrough MHC class I. Loss of surface MHC class I is classicallyassociated with NK-cell mediated cytolysis, however, NK cells can becomeexhausted within MHC I deficient tumors. As NK cells express the IL-18Rand our previous results in MC38 indicated that NK cells are expandedand activated by DR-IL-18, we thus tested whether DR-IL-18 couldstimulate NK cell attack against MHC I deficient tumors. We usedCRISPR/cas9 to knockout B2m in the Yummer1.7 cell line and found thatimplanted B2m-deficient YUMMER1.7 tumors were refractory to evencombined treatment with both anti-CTLA4 and anti-PD1 (FIG. 19A and FIG.19B), a combination that routinely cures close to 100% of parentalYummer1.7 tumors. However, single-agent treatment with DR-IL-18 (SEQ IDNO: 61) cured 60% of B2m-deficient Yummer1.7 tumors in an NK-celldependent fashion, as depletion with anti-NK1.1 abrogated the effect(FIG. 19A and FIG. 19B). Experiments were conducted to understand theeffect that DR-IL-18 had on intratumoral NK cells in the setting of anMHC class I deficient tumor. Immunophenotyping studies were performedwith flow cytometry on B2m-deficient Yummer.17 tumors from mice treatedwith saline or DR-IL-18. 24 hours after the 3rd dose of treatment, themice were sacrificed, tumors were dissociated, and the cell suspensionwas treated with PMA/ionomycin for four hours. The proliferative indexand functional capacity of the NK cells were then analyzed byintracellular flow cytometry with Ki67 and Interferon-gamma. As seen inFIG. 19C, NK cells from saline-treated B2m-deficient Yummer1.7 tumorshad scant Interferon-gamma production and Ki67 levels, indicating anexhausted phenotype. By contrast, NK cells from tumors treated withDR-IL-18 had robust Interferon-gamma production and Ki67 levels, withthe majority of NK cells being positive for both markers. These resultsthus establish that DR-IL-18 is effective in the treatment of MHC classI deficient tumors that are refractory to immune checkpoint blockade inan NK cell-dependent manner.

These results establish DR-IL-18 as a highly promising tumorimmunotherapeutic, and provide strong evidence that IL-18BP greatlylimits the effectiveness of IL-18 therapy, given the greatly improvedactivity of the SEQ ID NO: 61 DR-IL-18 variant. From these results, itis predicted that other strategies, such as blocking IL-18BP with anantibody, small protein, and/or small molecule could augment IL-18therapy and other immunotherapeutic regimens.

Efforts were undertaken to engineer an IL-18BP antagonist by creating a“decoy-to-the-decoy” (D2D), or IL-18 variants that specifically bindIL-18BP, but do not bind IL-18Rα and thus do not signal. The potentialadvantage of such an agent is that it would serve to neutralize IL-18BPand enhance the activity of endogenous IL-18, as opposed to drivingIL-18R signaling systemically. IL-18 was thus randomized at contactpositions for IL-18Rα (FIG. 20A) and a yeast-displayed library wasprepared as described previously for human and mouse DR-IL-18. Theresulting library of 3.9×10⁸ transformants was selected for 3 rounds asindicated in FIG. 20B, selecting for retained IL-18BP binding, whilecounter selecting against IL-18Rα. As seen in FIG. 20C, each round ofselection conferred enrichment for binding to IL-18BP (human and mouse),but without acquisition of IL-18Rα binding. 96 clones were sequenced,yielding 31 unique sequences, from which three consensus sequences SEQID NO: 123, SEQ ID NO: 124, and SEQ ID NO: 125 were derived (FIG. 21 ).Biophysical characterization of the resulting clones indicated that theyshowed similar binding isotherms to IL-18BP as WT IL-18 (FIG. 22A), butwith greatly decreased/absent binding to IL-18Rα (FIG. 22B). These dataare summarized in FIG. 22C. An identical selection process was performedfor murine IL-18, creating a library of 2.0×10⁸ transformants, which weselected to obtain 51 unique sequences summarized in FIG. 23 .

Example 2: Binding Affinity Measurements of Second Generation Variants

Surface Plasmon Resonance (SPR) was used to perform biophysical affinitymeasurements of second generation DR-IL-18 variants (binding to IL-18Rvs IL-18BP). Experiments were conducted using a Biacore T100 instrumentand carried out at 25° C. Biotinylated IL-18Rα or IL-18BP wereimmobilized onto a Biacore biotin capture chip (Series S CAP sensorchip, GE Healthcare) to yield an Rmax of ˜50 RU (IL-18Rα) or ˜10 RU(IL-18BP). Measurements were made with serial dilutions of the IL-18variants in HEPES buffered Saline-P+ buffer (10 mM HEPES pH 7.4, 150 mMNaCl, 0.005% surfactant P20). The surface was regenerated by three60-sec injections of regeneration buffer (3/4 (v/v) 8M guanidinehydrochloride with ¼ (v/v) 1M sodium hydroxide). Experiments wereperformed in multiple channels simultaneously for increasedobservations. All data were analyzed with the Biacore T100 evaluationsoftware version 2.0 with a 1:1 Langmuir binding model.

The results confirmed that DR-IL-18 variants of the disclosure exhibitgreatly reduced affinity for IL-18BP, and bind to IL-18Rα with at leastcomparable affinity as WT IL-18 (See FIG. 24 for the generatedsensograms, Table 10 for a summary of the measured kinetics, Table 11 isa summary of the affinity measurements, and Table 12 for a generalsummary, including results for the dissociation constant ratios of thesecond generation DR-IL-18 variants).

Table 10 is a summary of SPR data for second generation hDR-IL-18variants (kinetics)

Surface KD (M) KD (M) % Ligand Analyte ka (1/Ms) kd (1/s) Exp 2 Exp 1Rmax hIL- hIL-18 5.55E+05 2.97E−03 5.36E−09 5.35E−09 32 18Rα hIL- SEQ ID4.95E+05 9.10E−04 1.84E−09 2.24E−09 35 18Rα NO: 89 hIL- SEQ ID 6.31E+052.43E−03 3.85E−09 3.48E−09 35 18Rα NO: 90 hIL- SEQ ID 5.75E+05 1.19E−032.07E−09 2.65E−09 36 18Rα NO: 91 hIL- SEQ ID 2.18E+05 3.32E−03 1.52E−081.94E−08 19 18Rα NO: 87 hIL- hIL-18 5.18E+05 2.23E−07 4.30E−13 6.94E−1348 18BP hIL- SEQ ID Too weak to measure −1 18BP NO: 89 hIL- SEQ ID Tooweak to measure 2 18BP NO: 90 hIL- SEQ ID Too weak to measure 0 18BP NO:91 hIL- SEQ ID Too weak to measure −1 18BP NO: 87

Table 11 is a summary of SPR data for second generation hDR-IL-18variants (affinity)

KD_(apparent)hIL-18Rα KD_(apparent)hIL- Sample (nM) 18BP (nM) hIL-185.4, 5.4 <0.1 SEQ ID NO: 89 1.8, 2.2 too weak SEQ ID NO: 90 3.9, 3.5 tooweak SEQ ID NO: 91 2.1, 2.7 too weak SEQ ID NO: 87 15.2, 19.4 too weak

Table 12 is a summary of the SPR affinity measurements of secondgeneration hDR-IL-18 variants for IL-18Rα and IL-18BP. The IL-18 BP:RαDissociation Constant Ratio is the ratio of the KD for IL-18BP to the KDfor IL-18Rα normalized to the same ratio of WT IL-18. A higher numberfor this ratio indicates that the IL-18 variant has an enhancedpreference for binding IL-18Rα over IL-18BP compared to WT IL-18.*Average of 2 studies. k is a multiple of 1,000. m is a multiple of1,000,000.

IL-18 BP:Rα SPR: SPR: Dissociation K_(D) Rα K_(D)BP Constant Protein(nM) (nM) Ratio WT hIL-18 4.1*       0.002 1 SEQ ID NO: 34 8.0*     11.8* 3,024 SEQ ID NO: 36 9.1*      19.3* 4,348 SEQ ID NO: 37 7.7*    121*  32,215 SEQ ID NO: 89 2.2  >10,000 >9,318,275 SEQ ID NO: 903.5  >10,000 >5,857,201 SEQ ID NO: 91 2.7  >10,000 >7,592,669 SEQ ID NO:87 19.4   >10,000 >1,056,712 WT mIL-18 0.60        0.0011 1 SEQ ID NO:61 0.08   11,000 >75,000,136 A7, B1, C1, E8 0.22-1.7 14k-29k 9.3m-35m

Example 3: Efficacy for Cancer Treatment

Efficacy of DR-IL-18 variants was tested using multiple different cancermodels, including models of colorectal tumors, breast cancer, melanoma,and MHC class I deficient tumors that are resistant to immune checkpointinhibitors. The results show that DR-IL-18 variants with a bias to bindIL-18R and not IL-18BP can be used to treat a broad range of cancers(not limited to just those that were tested).

To efficacy of DR-IL-18 in a model of colorectal cancer, 250,000 CT26cells were implanted subcutaneously and treatment initiated at day 7once tumors were ˜60 mm³ on average. WT IL-18 and SEQ ID NO: 61 weredosed subcutaneously at 0.32 mg/kg twice weekly for a total of 5 doses.Anti-PD1 was given at 10 mg/kg at the same schedule. Only treatment withDR-IL-18, but not WT IL-18, resulted in tumor growth inhibition andtumor clearance in a subset of animals (FIG. 25A, providing an overlayof spider plots showing tumor growth of animals treated with saline(PBS, circles), WT IL-18 (squares), and DR-IL-18 (SEQ ID NO: 61,triangles). DR-IL-18, but not WT IL-18 resulted in prolonged survival(FIG. 25B, showing survival curves for mice treated with anti-PD-1, WTIL-18, or DR-IL-18 (SEQ ID NO: 61); numbers of complete responses areindicated in parentheses). 40% of mice treated with DR-IL-18 exhibitedtumor clearance, which was an improvement over the checkpoint inhibitoranti-PD-1. provides survival curves for mice treated with anti-PD-1, WTIL-18, and DR-IL-18 (SEQ ID NO: 61). Numbers of complete responses areindicated in parentheses. and tumor clearance in 40% of mice, animprovement over the checkpoint inhibitor anti-PD-1.

In both the in the 4T1 breast cancer model and B16-F10 melanoma model,only DR-IL-18, but not WT IL-18 resulted in tumor growth inhibition.Treatments were administered after tumors exceeded an average volume 50mm³ as indicated by the boxes marked with “t”.

The 4T1 breast cancer model and B16-F10 melanoma model were used toevaluate the efficacy of DR-IL-18 for additional cancer types. 500,0004T1 cells were implanted subcutaneously in BALB/C mice, and500,000B16-F10 cells were implanted subcutaneously in C57BL/6 mice.Treatments were administered after tumors exceeded an average volume 50mm³. In both models, only DR-IL-18, but not WT IL-18 resulted in tumorgrowth inhibition as shown in FIG. 26A (4T1 tumors) and FIG. 26B(B16-F10). Treatments were administered after tumors exceeded an averagevolume 50 mm³ as indicated by the boxes marked with “t”. The efficacy ofDR-IL-18 was also evaluated in MHC class I deficient tumor models. B2mdeficient (and thus MHC class I deficient) MC38 cells were preparedusing CRISPR/Cas9 mediated deletion as described for B2m deficientYUMMER cells. B2m−/− MC38 cells were implanted subcutaneously andtreatment initiated at day 7 once tumors were ˜65 mm³ on average. SEQ IDNO: 61 was dosed at 0.32 mg/kg twice weekly for 5 doses. Anti-PD1 andanti-CTLA4 were given at 8 mg/kg at the same schedule. The MHC class Ideficient tumors were resistant to immune checkpoint inhibition withanti-PD1 and anti-CTLA4, however DR-IL-18 treatment resulted in tumorgrowth inhibition (FIG. 27A).

RMA/S is a variant of the RMA lymphoma line that contains a spontaneousmutation in Tapasin, which results in a defect in antigen loading andtherefore decreased MHC class I surface expression. Congenic C57BL/6mice were implanted with 1,000,000 RMA/S cells subcutaneously andtreatment initiated at day 7. SEQ ID NO: 61 was dosed at 0.32 mg/kgtwice weekly. Anti-PD1 was given at 8 mg/kg at the same schedule. TheRMA/S tumors were resistant to immune checkpoint inhibition withanti-PD-1, while treatment with DR-IL-18 resulted in tumor growthinhibition (FIG. 27B).

Example 4: Promotion of Cancer Cell Killing by DR-IL-18 Alone or inCombination with a Therapeutic Antibody

An ex vivo cytotoxicity study was conducted to evaluate the ability ofDR-IL-18 to promote killing of cancer cells. CFSE labeled Raji (B celllymphoma) cells were used as target cells. Human i peripheral bloodmononuclear cells (PBMCs) were isolated and incubated with the CFSElabeled Raji cells \ at an effector:target (E:T) ratio of 1:10 for 25hours. The human DR-IL-18 variant hCS-1 (1 μM), the anti-CD20 antibodyrituximab (10 μg/mL), or the combination of both agents. Cytotoxicitywas measured by flow cytometry and calculated as the fraction of CFSEcells that became DAPI positive. DR-IL-18 treatment resulted in killingof target cells, and the combination of DR-IL-18 with rituximab resultedin a higher level of cancer cell killing than either agent alone,suggesting DR-IL-18 can enhance anti-tumor antibody-dependent cellmediated cytotoxicity (ADCC; FIG. 28 ; * p<0.05 by two-way ANOVA withTukey's correction for multiple comparisons). These data suggestDR-IL-18 can be combined with opsonizing agents, such as tumor-targetingantibodies, to enhance killing of cancer cells.

Example 5: Efficacy of DR-IL18 Against Viral Infections

This example demonstrates that DR-IL-18 can be used to treat infectiousdiseases, such as viral infections. C57BL/6 mice were infected with 106PFU of Vaccinia virus (VACV) intraperitoneally (IP) and administered 1mg/kg WT mIL-18 or DR-IL-18 (SEQ ID NO: 61) IP, as summarized in FIG.29A. Mice were sacrificed and viral titers were measured in the bloodand ovaries by RT-PCR on day 3 post-infection. Treatment with DR-IL-18,but not wild type mIL-18, resulted in a significant reduction in viralload in the blood and ovaries (FIG. 29B, * p<0.05, ** p<0.01, ***p<0.001). The efficacy of DR-IL-18 demonstrated in this model ofsystemic viral infection suggest that DR-IL-18 can be used to controlinfectious disease.

Example 6: Second Generation Human DR-IL-18 Variants Induce IL-18RSignaling

The ability of second generation human DR-IL-18 variants SEQ ID NO: 89,SEQ ID NO: 90, SEQ ID NO: 91, and SEQ ID NO: 87 to induce signaling viaIL-18 receptor was evaluated using IL-18 HEK-Blue reporter cells.Generation of these DR-IL-18 variants is described in Example 1, andtheir affinities for IL-18Rα and IL-18BP in Example 2.

HEK-Blue IL-18 sensor cells (InvivoGen) were maintained in completemedia (DMEM containing 10% heat-inactivated FBS, 2 mM L-glutamine, 50U/mL penicillin, and 501.1 g/mL streptomycin) supplemented with 100μg/mL Normocin, 30 μg/mL Blasticidin, 180 μg/mL Zeocin, and 200 μg/mLHygromycin. For cytokine activity measurements, 50,000 HEK-Blue IL-18sensor cells per well of a flat-bottom 96-well plate were incubated withrecombinant human IL-18 or the DR-IL-18 variants at successivelydecreasing concentrations in a total volume of 200 μL of complete media.After 24 hours of incubation at 37° C. and 5% CO₂, 30 μL of cell culturesupernatant was mixed with 170 μL QUANTI-Blue detection media(InvivoGen) and incubated at 37° C. and 5% CO₂ until a color change frompink to blue was detectable (0.5-4 hours). Levels of alkalinephosphatase were quantified using a spectrophotometer at 655 nmwavelength.

Human SEQ ID NO: 89, SEQ ID NO: 90, and SEQ ID NO: 91 showed enhancedpotency compared to WT hIL-18, whereas SEQ ID NO: 87 exhibitedapproximately equivalent potency as WT hIL-18 (FIG. 30 ). The datademonstrate, therefore, that all tested second generation human DR-IL-18variants actively signal through IL-18R.

Example 7: Cysteine Mutations to Enhance Stability of IL-18 Variants

SEQ ID NO: 89 is more stable than WT IL-18, however, it can formdisulfide-linked dimers, as well as intramolecular disulfide bonds.Thus, all combinations of single, double, triple, and quadruple cysteineto serine mutations were generated to test whether mutations at thesepositions could stabilize IL-18 molecules, and to determine whatcombination(s) of mutations provided the best stability.

Systematic mutation of all 4 cysteine residues to serine in SEQ ID NO:89 was performed as shown in Table 13.

Table 13 shows the tested cysteine variants of SEQ ID NO: 89.

Cys Cys Cys Cys Construct 38 68 76 127 SEQ ID NO: 89 C C C C SEQ ID NO:1 S S S S SEQ ID NO: 2 C S S S SEQ ID NO: 3 S C S S SEQ ID NO: 4 S S C SSEQ ID NO: 5 S S S C SEQ ID NO: 6 S S C C SEQ ID NO: 7 S C S C SEQ IDNO: 8 S C C S SEQ ID NO: 9 C S S C SEQ ID NO: 10 C S C S SEQ ID NO: 11 CC S S SEQ ID NO: 12 S C C C SEQ ID NO: 13 C S C C SEQ ID NO: 14 C C S CSEQ ID NO: 15 C C C S

Clones SEQ ID NOs: 1-15 (which are variants of SEQ ID NO: 89) weresynthesized by Twist and cloned into a Pichia pastoris expression vectorcontaining an n-terminal aga2 leader sequence for secretory expression.Plasmids were transformed into Pichia and expressed by methanolinduction at 30° C. for 60 hours. After induction, supernatants were runon an SDS/PAGE gel and visualized using Spyro-Ruby stain.

Clones SEQ ID NO: 5 and SEQ ID NO: 6 gave the best expression withoutevidence of intermolecular or intramolecular disulfide bond formation(FIG. 31 ; e.g., dimer band at −35 kDa in non-reduced samples and lowerdoublet band below 18 kD, respectively). These two clones contained thecombination of C38S and C68S mutations. Any clone with a C127 mutationexhibited lower expression yields. In FIG. 31 , R=Reduced sample;N=Non-reduced sample; MW=Molecular weight. The numbers along the bottomrefer to the SEQ ID number.

Conclusion: Mutations at positions C38 and C68 (see, e.g., SEQ ID NO: 6)provided the greatest degree of stabilization (e.g., in this case of theDR-IL-18 variant SEQ ID NO: 89). SEQ ID NO: 6 (C38S/C68S) inparticularly showed enhanced expression and greatly increased stability,albeit with slightly less (but nevertheless acceptable) potency.

Example 8: Removal of Predicted T Cell Epitope

The C68S mutation generated in Example 7 resulted in a predictedneo-epitope (e.g., T cell epitope). Thus, 6 additional non-immunogenicsubstitutions at position C68 (SEQ ID NO: 16-21) were constructed andtheir stability/potency were then tested.

Clones SEQ ID NO: 16-21 containing alternative substitutions (G, A, V,D, E, or N) at C68 were synthesized by Twist and cloned into an E. colicytoplasmic expression vector with an N-terminal 6×His-SUMO tag. E. coliwere grown in 100 mL Terrific Broth in shake flasks and induced withIPTG at 20° C. for 16 hours. After induction, proteins were purified byan immobilized metal affinity chromatography (IMAC) column; SUMO tagremoval was achieved by addition of his-tagged Ulp1 protease. Freehis-tagged SUMO and his-tagged Ulp1 were then removed by a second IMACcolumn. Yields and purity were assessed by SDS/PAGE.

The variants expressed at high titers (Table 14) and did not showevidence of disulfide bond formation by non-reducing SDS-PAGE gelanalysis (FIG. 32 ; R=Reduced sample; NR=Non-reduced sample).

Table 14 summarizes the additional mutations screened at positions 38and 68, corresponding sequences, and protein yields of the DR-IL-18variants.

Yield Variant (mg/L) Rank SEQ ID NO: SEQ ID NO: 6 76 7 6 (SEQ ID NO: 89plus C38S/C68S) SEQ ID NO: 16 115 1 16 (SEQ ID NO: 89 plus C38S/C68G)SEQ ID NO: 17 93 3 17 (SEQ ID NO: 89 plus C38S/C68A) SEQ ID NO: 18 79 618 (SEQ ID NO: 89 plus C38S/C68V) SEQ ID NO: 19 93 3 19 (SEQ ID NO: 89plus C38S/C68D) SEQ ID NO: 20 91 5 20 (SEQ ID NO: 89 plus C38S/C68E) SEQID NO: 21 106 2 21 (SEQ ID NO: 89 plus C38S/C68N)

As shown in FIG. 33 , An accelerated “shelf stability” study wasperformed to further evaluate the stability of DR-IL-18 variants.Protein preparations were formulated in PBS at 1 mg/mL and held at 51°C. for 48 hours. Samples were taken at several timepoints, filteredthrough a 0.45 μm filter, and analyzed by size-exclusion chromatography(SEC). The DR-IL-18 variants exhibited a range of stabilities underthese conditions, with SEQ ID NO: 16, SEQ ID NO: 19, and SEQ ID NO: 17showing the greatest retention in the % of the main peak relative to t=0(FIG. 33 ). The activity of WT IL-18 versus the DR-IL-18 variants wasmeasured using an IL-18 reporter assay in which IL-18 receptor signalingresults in expression of secreted alkaline phosphatase (HEK-Blue IL-18from InVivoGen). Reporter cells were cultured, stimulated, and secretedalkaline phosphatase levels measured, as described in Example 6. Allvariants showed increased potency of IL-18R signaling induction comparedto WT IL-18, and similar potency to the parent variant SEQ ID NO: 89(Table 15 and FIG. 34 ).

Table 15 shows EC50 values of DR-IL18 variants for inducing IL-18Rsignaling as determined by a HEK-Blue reporter cell line assay (see alsoFIG. 34 )

Variant EC50-#1 (M) EC50-#2 (M) Rank SEQ ID NO: hIL-18 WT 1.45 × 10⁻⁹ 1.16 × 10⁻⁹  — 30 SEQ ID NO: 89 1.43 × 10⁻¹⁰ 5.44 × 10⁻¹¹ 2 89 SEQ IDNO: 6 1.96 × 10⁻¹⁰ 1.30 × 10⁻¹⁰ 7  6 SEQ ID NO: 16 2.05 × 10⁻¹⁰ 1.54 ×10⁻¹⁰ 8 16 SEQ ID NO: 17 1.41 × 10⁻¹⁰ 1.09 × 10⁻¹⁰ 4 17 SEQ ID NO: 185.19 × 10⁻¹¹ 4.48 × 10⁻¹¹ 1 18 SEQ ID NO: 19 1.24 × 10⁻¹⁰ 7.90 × 10⁻¹¹ 319 SEQ ID NO: 20 1.35 × 10⁻¹⁰ 1.62 × 10⁻¹⁰ 6 20 SEQ ID NO: 21 1.28 ×10⁻¹⁰ 1.24 × 10⁻¹⁰ 5 21

Table 16 summarizes the rankings of yield, potency, and stability ofDR-IL-18 variants determined by the assays in this example

Stability SEQ (51° C. ID Variant Yield Potency 48 h) NO: SEQ ID NO: 89 82 6 89 (SEQ ID NO: 89 plus C38S/C68S) 7 7 4  6 (SEQ ID NO: 89 plusC38S/C68G) 1 8 1 16 (SEQ ID NO: 89 plus C38S/C68A) 3 4 12  17 (SEQ IDNO: 89 plus C38S/C68V) 6 1 8 18 (SEQ ID NO: 89 plus C38S/C68D) 3 3 2 19(SEQ ID NO: 89 plus C38S/C68E) 5 6 7 20 (SEQ ID NO: 89 plus C38S/C68N) 25 14  21

Example 9: Characterization of Stability, Affinity, and Potency of aDR-IL-18 of the Disclosure

SEQ ID NO: 19 was selected for further characterization based on itsfavorable profile of potency, stability, and immunogenicity (seeExamples 7 & 8). Additional assays were performed to compare stability,affinity, and potency of SEQ ID NO: 19 (C38S/C68D) to SEQ ID NO: 89 (theparent C38/C68 molecule), and to wild type human IL-18.

The effect of freeze thaw cycles on SEQ ID NO: 19 and SEQ ID NO: 89 wastested. The proteins were formulated in 20 mM acetate pH 5.0, 8%sucrose, 0.1 mM EDTA, and 0.02% Polysorbate (PS) 80. The formulationswere then frozen at −80° C. and thawed at 25° C. five times. Theproteins were then analyzed by size-exclusion chromatography. A decreasein main peak of SEQ ID NO: 89 was observed, as was an appearance of adimer peak (indicated by arrow) (FIG. 35 , upper panel). In contrast, noor minimal changes were detected for SEQ ID NO: 19 (FIG. 35 , lowerpanel), demonstrating superior stability of this DR-IL-18 variant afterexposure to freeze-thaw cycles.

A stability comparison using an agitation study was performed afterexposure to agitation at 37° C. SEQ ID NO: 19 and SEQ ID NO: 89 proteinswere formulated in 20 mM acetate, pH 5.0, 8% sucrose, 0.1 mM EDTA, and0.02% PS80. The samples were agitated with shaking at 37° C. for 72hours. Samples were taken at t=0, 24 (dl), and 72 (d3) hours andanalyzed by size exclusion chromatography. A decrease in main peak ofSEQ ID NO: 89 was observed, as was a relative increase in dimer/HMW(high molecular weight) species indicated by arrows (FIG. 36 , upperpanel). In contrast, no or minimal changes were detected for SEQ ID NO:19. FIG. 36 depicts size-exclusion chromatography data comparing SEQ IDNO: 19 to SEQ ID NO: 89 from the agitation study.

IL-18 receptor affinity. The affinities of human IL-18 and SEQ ID NO: 19were determined for both human and cynomolgus monkey IL-18Rα and IL-18BPusing surface plasmon resonance. Biotinylated receptor proteins wereimmobilized on a streptavidin capture chip and IL-18 or SEQ ID NO: 19were applied as the free analyte. Single-cell kinetics were used todetermine binding. WT hIL-18 and SEQ ID NO: 19 had similar bindingaffinity for IL-18Rα in this assay (FIG. 37A). However, WT IL-18 boundIL-18BP with extremely high affinity (<1 nM), whereas SEQ ID NO: 19exhibited no detectable binding to IL-18BP (FIG. 37B).

The activity of WT IL-18 and SEQ ID NO: 19 in the presence of IL-18BPwas evaluated using an IL-18 reporter assay in which IL-18 receptorsignaling results in expression of secreted alkaline phosphatase(HEK-Blue IL-18 from InVivoGen). Reporter cells were cultured andsecreted alkaline phosphatase levels measured as described in Example 6,except that WT IL-18 or SEQ ID NO: 19 were applied to HEK-BLUE IL-18reporter cells at a fixed concentration of 1.0 or 0.1 ng/mL,respectively, in the presence of a range of IL-18BP concentrations, from10⁻⁶ to 10⁻⁹ M. Downstream secreted embryonic alkaline phosphatase(SEAP) activity was measured according to the manufacturer'sinstructions. WT hIL-18 was potently inhibited by addition of IL-18BP,whereas SEQ ID NO: 19 retained strong induction of IL-18R signaling atall concentrations of IL-18BP (FIG. 38 ).

Example 10: Dosing of IL-18 Polypeptides

A first dosing study was performed to compare efficacy and tolerabilityof dosing with DR-IL-18 once per day, every other day, two times perweek, and 1 time per week in a cancer model This study was carried outin mice, and therefore used SEQ ID NO: 61, which is a mouse DR IL-18variant polypeptide (i.e., a mouse IL-18 that binds to and activatesmouse IL-18R but is not inhibited by mouse IL-18BP). The human variantswere not used because the human IL-18 protein does not cross-react withmouse IL-18R (i.e., it does not bind to and active mouse IL-18R).

250,000 MC38 cells were implanted subcutaneously and treatment wasinitiated once tumors were at 80 mm³. Dosing was qD (once daily), qoD(once every other day), BiW (Twice Weekly,), or qW (Once Weekly), givensubcutaneously for 2 weeks. All doses and schedules showed efficacy(FIG. 39 ). Most regimens were well tolerated. However, daily dosingshowed toxicity at 0.1 mpk (mg/kg) and higher. Tumor outgrowth generallyoccurred after treatment was discontinued. Tumor growth curves aredepicted with the last point carried forward once endpoint was reached.Curves were terminated once 50% or more animals reached endpoint.Results: Daily dosing at 0.1 mpk (mg/kg) and higher appeared to be toxicand was associated with weight-loss. DR-IL-18 was effective at all otherdosing schedules. Efficacy was superior to anti-PD-1 therapy, which wasevident at all doses tested and even at once-weekly dosing.

A second dosing experiment was performed to compare dosing once per weekversus twice per week in a cancer model. As above, SEQ ID NO: 61 wasused because these studies were carried out in mice. 250,000 MC38 cellswere implanted subcutaneously and treatment was initiated once tumorsreached 100 mm³ (day 7). Animals were administered four doses at afrequency of once per week (qW) or two doses at a frequency of onceevery two weeks (q2W) by subcutaneous injection. A 100-fold range ofdoses (from 0.01 to 1.0 mg/kg) was tested. All doses and schedulesshowed efficacy, even at 0.01 mg/kg. The maximum effective dose (MaxED)was about 0.32 mg/kg at both qW and q2W dosing (FIG. 40 ). Strong DRIL-18 efficacy was observed even with infrequent pulse-dosing. ven onceevery 2 week dosing was more effective that twice per week anti-PD-1treatment. This was very surprising given the dosing requirements ofother interleukins, (e.g., IL-2/IL-15, IL-10) that require half-lifeextension or frequent dosing (e.g., TID dosing of IL-2).

Monkey tolerability assays were performed to determine how welltolerated DR-IL-18 was at various doses, and using once weekly (qW) ortwice weekly (BiW) dosing schedules. Cynomolgus macques were treatedwith DR-IL-18 (SEQ ID NO: 89 or SEQ ID NO: 19) subcutaneously, weekly(qW) or twice weekly (BiW), with doses ranging from 0.001 mg/kg to 3.2mg/kg. Blood was drawn periodically and hemoglobin levels in the bloodmeasured. An illustrative dosing and sampling regimen is provided inFIG. 41A. The results showed dosing frequencies more than 1×/weekelicited undesirable decreases in hemoglobin concentrations in thestudy. Cynomolgus macques treated with SEQ ID NO: 89 subcutaneouslytwice per week exhibited a dose-dependent reduction in hemoglobinrelative to saline-treated monkeys (FIG. 41B, upper panel). By contrast,once weekly treatment with DR-18 (SEQ ID NO: 89 or SEQ ID NO: 19), evenup to 3.2 mg/kg, did not result in decreased hemoglobin levels relativeto control saline treatment (FIG. 41B, upper panel and lower panel).

Example 11: Producing DR-IL-18 Variant in Bacteria (E. coli)

Process 1: Cell-Free In Vitro Cleavage with SUMO-Protease

In this example, DR-IL-18 (SEQ ID NO: 89 in this case) was tagged with aHis-tagged SUMO tag, expressed in E. coli, and a His-tagged SUMOprotease was used to cleave the SUMO tag, producing an active IL-18protein.

DR-IL-18 was cloned into an E. coli cytoplasmic expression vector withan N-terminal 6×His-SUMO tag. E. coli were grown in 100 mL TerrificBroth in shake flasks and induced with IPTG at 20° C. for 16 hours.Initial capture of the fusion protein (His-tagged SUMO tag fused to theDR-IL-18) from bacterial lysate was performed using immobilized metalaffinity chromatography (IMAC). This was efficient, with high step-yieldat >90% purity. After the cleavage reaction with His-tagged Ulp1 SUMOprotease to remove the SUMO tag, the removed His-tagged SUMO tag and theHis-tagged SUMO protease were efficiently removed using IMAC indepletion mode. Yields and purity were assessed by SDS/PAGE. Theresulting protein was >90% pure after this step (often >95%) (FIG. 42A).Cleavage was efficient and can be conducted at ratios of 1:500 orgreater. Note: Expression at lower temperature (21° C.) greatlyincreases soluble yields (this particular study was performed at 26°C.). FIG. 42B summarizes the processing and purification steps.

FIG. 43A and FIG. 43B provide results of the cleavage reaction, asmonitored by RP-HPLC assays. The RP-HPLC assay can be used to calculatestep-yield and titers, as well.

Process 2: Cleavage Inside of a Bacterial Cell with Co-ExpressedSUMO-Protease

This example demonstrates that SUMO tagged IL-18 (in this case thestabilized DR IL-18 SEQ ID NO: 19) and SUMO-protease can be co-expressedin bacteria (in this case E. coli), resulting in removal of the SUMO tagvia cleavage inside of the bacterial cells. The SUMO protease wasexpressed under control of a rhamnose-inducible promoter. Rhamnoseinduction of the protease allowed for separate timing and tunableinduction of the protease relative to the IL-18 (e.g., DR-IL-18). E.coli expressing the SUMO-tagged DR-IL-18 were treated with 0.1 to 2 mML-rhamnose, resulting in induction of SUMO protease expression, andcleavage of the SUMO tag to liberate untagged SEQ ID NO: 19 (FIG. 44 ).Note: On a non-reducing gel, the SUMO tag runs above DR-18; somecleavage can be observed without rhamnose treatment due to promoter“leakiness”.

Total expression of the cleaved product was comparable to theSUMO-fusion (after accounting for SUMO mass). In vivo (in bacterialcells) cleavage was monitored using RP-HPLC (FIG. 45 , showing cleavageof the SUMO tag for rhamnose-treated cultures). 0.2-0.5 mM L-rhamnoseappeared to provide good results in these particular experiments.

Successful results have been obtained by placing SUMO-proteasedownstream of the IL-18 (e.g., DR-IL-18) in a bicistronic format, aswell as by expressing the SUMO protease from a separate promoter (e.g.,from a separate plasmid), e.g., under an inducible promoter such as therhamnose-inducible promoter, as in this example.

A downstream process can be used to capture and purify the cleavedproduct (i.e., the active DR-IL-18 protein) (see, e.g., Example 12).

In vivo (in bacterial cells) cleavage was monitored using RP-HPLC. FIG.45 provides the chromatographs for in vivo (in bacterial cells)cleavage. Note: “Leakiness” of the rhamnose promotor created enough SUMOprotease to cleave most of the fusion protein. 0.2-0.5 mM L-rhamnoseappeared to be the optimal concentration range in these particularexperiments.

Example 12: Producing IL-18 Variant Using Yeast (P. pastoris)

A system for expression and secretion of DR-IL-18 was developed usingPichia Pastoris (yeast). A signal peptide was added to the DR-IL-18 andcloned into an expression vector (FIG. 46 , top). This system results insecretion of the DR-IL-18 and cleavage of the signal peptide by SignalPeptidase/Kex2, avoiding the need for a protease to remove a tag.

In this illustrative example, the DR-IL-18 variants SEQ ID NO: 89 andSEQ ID NO:87 were tested. The DR-IL-18 variants were synthesized byTwist Biosciences and cloned into a Pichia pastoris expression vectorcontaining an n-terminal aga2 leader sequence for secretory expression.Plasmids were transformed into Pichia and expressed by methanolinduction at 30° C. for 60 hours, resulting in production of DR-IL-18 athigh titers, including >1 g/L in some cases. DR-IL-18 with the activenative N-terminus (tyrosine in the case of human IL-18) was directlysecreted into the media (FIG. 47 ).

A 2-chromatographic-step process was used for tagless purification ofIL-18 (in this case the DR IL-18 SEQ ID NO: 89) to >97% purity, 96%monodispersity, <2 EU/mg (FIG. 46 and FIG. 48 ; UFDF=Ultrafiltration(UF) and diafiltration (DF). “Capto MMC” is a multimodal salt-tolerantresin for capture and intermediate purification of proteins from largefeed volumes by packed bed chromatography. HIC=Hydrophobic InteractionChromatography (e.g., depicted in this figure is phenyl sepharose HIC).

DR-18 purification was performed. FIG. 48 shows SDS-PAGE gel showingpurification of DR-18 (e.g., from P. Pastoris or from bacteria). A2-chromatographic-step process facilitated tagless purification of IL-18(in this case the DR IL-18 SEQ ID NO: 89) to >97% purity, 96%monodispersity, <2 EU/mg. UFDF=Ultrafiltration (UF) and diafiltration(DF). “Capto MMC” is a multimodal salt-tolerant resin for capture andintermediate purification of proteins from large feed volumes by packedbed chromatography. HIC=Hydrophobic Interaction Chromatography (e.g.,depicted in this figure is phenyl sepharose HIC).

Example 13: Generation of a Cell Bank for Production of a DR IL-18Variant

This example demonstrates generation of a plasmid system and cell bankfor production of SEQ ID NO: 19, a DR IL-18 variant of the disclosurewith mutations at positions C38 and C68.

A dual plasmid system was generated for expression of SEQ ID NO: 19 inE. coli. For full activity, IL-18 (e.g., DR IL-18, such as SEQ ID NO:19) can require its native N-terminus rather than an N-terminalmethionine. Accordingly, a plasmid was designed using a smallubiquitin-related modifier (SUMO) expression system which utilizes aSUMO leader sequence from Saccharomyces cerevisiae at the N-terminus,such that the SUMO leader can be cleaved off by the SUMO protease, ULP1.

Expression of the DR IL-18 with SUMO leader peptide was driven by a T7promotor in a high copy plasmid to maximize expression. A pD451-SRplasmid backbone was used. This plasmid contains a kanamycin resistancegene, a pUC origin of replication, and has the T7 promotor present toexpress the SEQ ID NO: 19 protein after isopropyl(3-d-1-thiogalactopyranoside (IPTG) induction.

A second lower copy number plasmid was used for expression of the SUMOprotease driven by a rhamnose promotor, which is relatively weak, suchthat expression can be tuned by varying the amount of rhamnose added tothe culture. The SUMO protease gene was cloned into the plasmidpD883-SR. This plasmid contains a chloramphenicol resistance gene, ap15a origin of replication, and has the rhamnose promotor as part of therhaBAD regulatory sequence to enable controlled expression of the SUMOprotease after rhamnose induction.

Both plasmids were transformed simultaneously into the E. coli strain T7Express, and clones with the plasmids were selected using agar platescontaining both kanamycin and chloramphenicol. The T7 Express is astrain that allows for very high expression of genes utilizing the T7promotor after IPTG induction. It contains the T7 polymerase in thelactose operon and contains no lambda prophage. The system of twoplasmids with different origins of replication and different antibioticresistance genes allows both plasmids to be maintained in a stablemanner and tested for independently. The SUMO protease and the SUMOleader peptide both have N-terminal His-tags, thereby allowing theirremoval by immobilized metal affinity chromatography. This expressionsystem was designed to allow very high expression of correctly processedSEQ ID NO: 19 that has been properly cleaved by the SUMO protease, forexample, at 2-3 g/L.

E. coli strain T7 Express competent cells were transformed with 1 μL ofeach of the two plasmids. The transformed cells were grown overnight onLB agar plates supplemented with 50 μg/mL of kanamycin (KAM) and 50μg/mL of chloramphenicol (CAM) at 37° C. A single, well grown colony,was inoculated into 10 mL of medium and grown overnight at 30° C. A cellbank was prepared by mixing equal volumes of overnight culture with 30%(v/v) sterile glycerol solution, then the mixed solution was aliquotedinto suitable vials (at 200 μL), and stored at −20° C.

One vial was thawed, subcultured onto TBA (Terrific Broth Agar) platescontaining 50 μg/mL of KAM and 50 μg/mL of CAM, and incubated at 30° C.for 20 to 24 hours. A single colony was inoculated into 50 mL of mediumand incubated in a shaker-incubator overnight at 30° C., 220 rpm. Thisculture was inoculated at 0.2 to 0.3 OU at OD600 nm and incubated in ashaker incubator at 30° C., 220 rpm. The final culture OD600 nm was 1.9(before glycerol). A new cell bank was prepared by mixing of 56 mL ofcell culture with 8 mL of 80% sterile glycerol (10% final concentration)and aliquoting into 50 cryo-vials (1 mL each). The vials were labeledand stored directly in a −80° C. freezer. This cell bank was testedbefore and after freezing, demonstrating stability of the plasmids andretention of a high concentration of viable cells (Table 17).

TABLE 17 Viability, purity, and plasmid stability of cell bank samplesbefore and after freezing Before freezing After freezing Viable cellcount 7.7 × 10{circumflex over ( )}8 CFU/mL 7.3 × 10{circumflex over( )}8 CFU/mL Purity Conforms Conforms Plasmid stability 98% 97%

One vial was gently thawed at 2° C. to 8° C. for ≤30 min. After thawing,1 mL of cell suspension was transferred into a 0.5 L shake flask withprepared TB medium with added salt and antibiotics (kanamycin andchloramphenicol). This culture was incubated for 15 to 18 h at 37° C.with an agitation rate of 300 rpm until target optical density of ≥5.0OU was reached. After that, the required amount of cell suspension fromthe first growth was transferred to a 1 L shake flask for furthercultivation for at least for 3 to 4 hours until a required opticaldensity of 1.50 OU to 3.50 OU was reached. 160 mL of the second growthcell culture was combined and mixed with 160 mL of 50% glycerol solutionand 1.0 mL of cell suspension was aliquoted into sterile cryogenic vialsand labelled appropriately. Vials from this second cell bank were frozenat −80±10° C. In-process controls of the cell bank preparation wereculture purity after freezing, total viable cell count after freezing,and plasmid stability after freezing. The tests showed that nobacteriophage contamination was present, and a a plasmid copy number of288.15. The IL-18 encoding plasmid retained 100% sequence identity tothe originally designed sequences, and restriction enzyme digestionyielded expected plasmid fragment sizes indicating no changes to theplasmid structure or insert sequences.

Example 14: Ellman's Titration for Free Thiols

This example demonstrates that native SEQ ID NO: 19, a DR IL-18 variantof the disclosure with mutations at positions C38 and C68, contains verylittle free cysteine.

Ellman's Reagent (5,5′-dithio-bis-[2-nitrobenzoic acid]/DTNB) was usedto estimate sulfhydryl groups in a sample by comparing to a standardcurve of a sulfhydryl-containing compound. DTNB, upon reaction with freethiols of proteins, generates a colored product, 2-nitro-5-thiobenzoate(TNB) that absorbs at 412 nm. For every mole of thiol that reacts withDTNB, one mole of TNB is produced, and by quantifying the amount of TNB,the number of free cysteines present per mole of protein can beestimated. The results of the assay show that very little free cysteineis present in native SEQ ID NO: 19 (Table 18), consistent with mutationof C38 and C68 to other residues, a lack of solvent exposure of the tworemaining cysteines in SEQ ID NO: 19, and reduced formation ofintramolecular and intermolecular disulfide bonds observed forpolypeptides with such mutations in Examples 7 and 8.

TABLE 18 Free thiol group determination in samples of SEQ ID NO: 19.Free thiol Protein, groups, Protein, Avg mol/ mol/protein, CV, mol/freeSample intensity sample mol SD % thiol, mol RS, dilution 3804.5  1.8E−080.0041 71.4 1.88 242.96 −3.0 RS, dilution 5756.5 2.69E−08 0.0042 154.92.69 239.15 −2.0 RS, dilution 7220.5 3.59E−08 0.0039 143.5 1.99 253.50−1.5 Mean 0.0041 245.20

Example 15: Formulation Development

Formulation development was performed to select drug substance and drugproduct formulation for toxicology and clinical studies. The SEQ ID NO:19 drug substance formulation development process included evaluation ofoptimal pH, protein concentration, and excipient screening. Formulationwas intended to support the following product attributes: liquid form,sterility, suitability for subcutaneous injection, and a shelf life ofat least 24 months at 2° C. to 8° C. or −20° C. temperature.Formulations were assessed through stability studies. The details ofstability studies are mentioned below:

-   -   1. Stressed samples were compared to initial samples for        possible alterations of protein integrity at selected pH value.        Stress conditions were 40±2° C./25% RH for up to 3 weeks.    -   2. Freeze-thaw cycles were performed to assess sensitivity of        the protein to repeated freezing and thawing. Up to five        freeze-thaw cycles were performed at the following conditions:        −80° C. for up to 16 hours, then at room temperature for 8        hours.    -   3. Agitation with shaking was performed to assess protein        degradation pathways and rates (25° C./60% RH for up to 2 weeks        at 150 rpm). SEQ ID NO: 19 drug substance formulation        development studies are summarized in Table 19.

TABLE 19 Protein Material Formulation concentration batch StabilityQuality Step Composition pH (mg/mL) No. study types attributes Study No.1 Selection 10 mM L- 6 10 P57- Stressed Fragments/ of an Histidine/L- 20BTPH- conditions soluble optimal Histidine-HCl, 240 6.5 15 097- (40degrees aggregates pH mM Sucrose, 0.2% 20 2004M- C.) for 1 and by SE-(w/v) Polysorbate 7 10 262 3 weeks HPLC and 20, pH 6.0 20 non- reducingSDS-PAGE Protein concentration measured at A280 Oxidation by RP- HPLCStudy No. 2 Lead 10 mM His/His- 6.5 30 P57- Accelerated Proteinformulation HCl, 8% sucrose, BTPH- conditions concentration selection0.1 mM EDTA, 5 097- (25 degrees turbidity, mM L-Methionine, 2006M-Celsius) measured at 0.02% 402 3. Freeze- A280, (w/v) Polysorbate thaw 5A340/A4350 80 cycles Charge 10 mM L- 6.5 20 isoforms by Histidine/L-AEX-HPLC Histidine-HCl, 8% Oxidation Sucrose, 5 mM L- by RP- Methionine,0.1 HPLC mM EDTA, 0.02% Sub-visible w/v Polysorbate 80 particles by 10mM L- 7 30 MFI Histidine/L- Histidine-HCl, 8% Sucrose, 5 mM L-Methionine, 0.1 mM EDTA, 0.02% w/v Polysorbate 80 Abbreviations:AEX-HPLC = anion exchange liquid chromatography; MFI = micro-flowimaging; RP-HPLC = reverse phase high performance liquid chromatography;PS80—polysorbate 80; SDS-PAGE = sodium dodecyl sulphate polyacrylamidegel electrophoresis; SE-HPLC = size exclusion liquid chromatography.

During Study 1, six formulations with different concentration and pHwere compared at thermal stress conditions. Formulations with lower pHwere less stable after 3 weeks at 40±2° C./25% RH. Thus, pH 6.5 and 7.0were chosen for Study 2. Also, protein purity during Study 1 did notshow a dependency on protein concentration. Thus, protein concentrationsof 20 mg/mL and 30 mg/mL were used in Study 2. During Study 2,additional excipients were added to the formulations,ethylenediaminetetraacetic acid and L-methionine, to maintain optimalprotein stability and prevent its oxidation. No differences between theformulations were observed at all tested conditions. According to theformulation development results, the final selected composition was 30mg/mL of SEQ ID NO: 19 protein in 10 mM L-histidine/L-histidine-HCl, 8%sucrose, 0.02% (w/v) polysorbate 80 at pH-6.5.

Example 16: Human Administration to Determine Treatment Dose

The primary objective of this study is to determine the MTD and the RP2Dof SEQ ID NO: 19 in patients with relapsed or refractory solid tumors.The secondary objectives are to evaluate the overall safety andtolerability of SEQ ID NO: 19 and to characterize the pharmacokinetic(PK) profile of SEQ ID NO: 19 as a single agent. Investigators monitorthe safety of the study by looking at: the incidence of DLTs and adverseeffects (AEs), changes in clinical laboratory parameters, vital signs,ECG testing and parameters, physical examination, and incidence ofanti-SEQ ID NO: 19 antibodies. Investigators additionally monitor thepossibility of patients developing cytokine storms, since SEQ ID NO: 19is an immune agonist.

Patients with relapsed or refractory solid tumors are enrolled in a doseescalation study to determine the maximum tolerated dose (MTD) and therecommended Phase 2 dose (RP2D) of SEQ ID NO: 19. Eligible patients are18 years or older and diagnosed with solid tumors that have diseaseprogression through standard therapy or for whom standard of caretherapy that prolongs survival is unavailable or unsuitable. Patientsreceive a subcutaneous (SC) dose of SEQ ID NO: 19 once a week for 28days (one cycle). The dose can range from 30 μg of SEQ ID NO: 19 per kgof patient (μg/kg) to 1200 μg/kg. Further dose escalations can includeranges from 1200 μg/kg to 1.5 mg/kg. The initial testing dose is 30μg/kg. Any dose within those two ranges is testable within the study todetermine the MTD and RP2D. Dose escalation follows an mTPI model withcohorts of 2 patients initially with a primary dose limiting toxicities(DLT) monitoring period of 28 days following the first dose. Patientsare monitored for 7 days after the first dose of a new dose before newpatients may receive that new dose amount.

The mTPI model uses a simple beta-binomial hierarchical model, where thedecision rules are based on calculating the unit probability mass (UPM)of 3 dosing intervals corresponding to under, proper, and over-dosing.The mTPI method calculates the UPMs for the 3 dosing intervals, and theone with the largest UPM implies the corresponding dose-finding decisionand that decision provides the dose level to be used for futurepatients. The target toxicity probability rate is 25% with an acceptableDLT interval of 20-30%. If the initial two patients in the first cycleexperience no DLTs then the enrollment of the next cohort begins at thenext dose level in the next cycle. If one of the two initial patientsexperience a DLT, then the dose will be lowered to the next lowest dose.If no additional DLTs are observed at the lower dose level when thecohort expands to 6 patients, dose escalation will be resumed. Once thefirst DLT is observed, only ≤33% dose increments will be applied to allthe remaining cohorts.

The estimation of the MTD at the end of study derives from isotonicregression. Specifically, the MTD estimate is the dose level whichisotonic estimate of DLT rates is less than or equal to 25% and it isthe dose level for which the isotonic estimate of the DLT rate isclosest to the target DLT rate. Dose escalation is stopped if themaximum sample size of 42 patients has been achieved, 6 to 12 patientshave been enrolled at a dose level that is predicted to be the MTD, orall doses explored appear to be overly toxic and the MTD cannot bedetermined.

Blood samples for PK analysis are collected from all and investigatorsuse these samples to calculate the PK parameters. Blood samples arecollected on days 1, 2, 3, 8, 15, and 22 of cycle 1, days 1 and 15 ofcycle 2, and on day 1 of alternate cycles staring with cycle 3 untilstudy completion. Patients also provide a blood sample 30 days after thelast dose of SEQ ID NO: 19 and before the patients start anew therapy.PK parameters C_(max), t_(max), AUC₀₋₁, AUC_(τ), AUC_(0-∞), and t_(1/2)are estimated from plasma concentration-time data usingnon-compartmental analysis if possible. Additional PK parameters may beconsidered if appropriate. Investigators also monitor patients are thedevelopment of anti-drug antibodies using the blood samples.

Example 17: Efficacy Testing in Patients with Solid Tumors

The primary objective of this study is to evaluate the anti-tumoractivity of SEQ ID NO: 19 at the RP2D in relapsed solid tumor cohorts.The primary efficacy endpoint for this study is confirmed objectiveresponse looking at patients that have a complete response (CR) orpartial response (PR) by RECIST 1.1. Secondary efficacy endpoints willinclude: best objective response, disease control rate (DCR) (CR, PR, orstable disease (SD) for >12 weeks), duration of response (DOR), time toresponse (ToR), progression free survival (PFS), and overall survival(OS).

Patients with distinct solid tumor types are enrolled in a study toevaluate the efficiency of SEQ ID NO: 19 treatment in these tumor types.Eligible patients are 18 years or older and have one the of followingsolid tumor types: melanoma, renal cell carcinoma (RCC), triple-negativebreast cancer (TNBC), non-small cell lung cancer (NSCLC), squamous cellcarcinoma of the head and neck (SCCHN), or Microsatellite Instable High(MSI-Hi) tumors. Within 28 days of the first dose, patients undergo amandatory tumor biopsy. For RCC, TNBC, NSCLC, and MSI-Hi, up to 25patients may be enrolled for each tumor type; metastatic melanoma andSCCHN cancer types can enroll up to 28 patients per cancer type. Ifinvestigators do not observe any objective response in a cancer typecohort, then no additional patients will be enrolled into that cohort.

Patients receive one subcutaneous dose of SEQ ID NO: 19 once a week for28 days (one cycle). The dose is the RP2D from the human dose escalationstudy. Patients undergo disease assessments every 8 or 12 weeks duringthe study and may include computed tomography (CT) scans, magneticresonance imaging (MRI) scans, and/or positron emission tomography (PET)scans. Investigators evaluate all target and nontarget lesions from thescans using Response Evaluation Criteria in Solid Tumors (RECIST 1.1).Prior to the start of cycle 2, patients undergo a second mandatory tumorbiopsy. If disease progression occurs while in the study, patients mayrequest an optional biopsy at the time of disease progression.

Blood samples for PK analysis are collected from all patients andinvestigators use these samples to calculate the PK parameters. Bloodsamples are collected on days 1 and 15 of cycle 1, days 1 and 15 ofcycle 2, and on day 1 of alternate cycles staring with cycle 3 untilstudy completion. Patients also provide a blood sample 30 days after thelast dose of SEQ ID NO: 19 and before the patients start a new therapy.PK parameters C_(max), t_(max), AUC₀₋₁, AUC_(τ), AUC_(0-∞), and t_(1/2)are estimated from plasma concentration-time data usingnon-compartmental analysis if possible. Additional PK parameters may beconsidered if appropriate. Investigators also monitor patients are thedevelopment of anti-drug antibodies using the blood samples.

Amino Acid Sequences

Wild-type IL-18 amino acid sequences HUMAN Interleukin-18 (mature form)(SEQ ID NO: 30) YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDR SIMFTVQNEDMOUSE Interleukin-18 (mature form) (SEQ ID NO: 31)NFGRLHCTTAVIRNINDQVLFVDKRQPVFEDMTDIDQSASEPQTRLIIYMYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFQKRVPGHNKMEFESSLYEGHFLACQKEDDAFKLILKKKDENGDKSV MFTLTNLHQS Q14116|IL18_HUMAN Interleukin-18 (uncleaved precursor) (SEQ ID NO: 32)MAAEPVEDNCINFVAMKFIDNTLYFIAEDDENLESDYFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNEDP70380|IL18_MOUSE Interleukin-18 (uncleaved precursor) (SEQ ID NO: 33)MAAMSEDSCVNFKEMMFIDNTLYFIPEENGDLESDNFGRLHCTTAVIRNINDQVLFVDKRQPVFEDMTDIDQSASEPQTRLIIYMYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFQKRVPGHNKMEFESSLYEGHFLACQKEDDAFKLILKKKDENGDKSVMFTLTNLHQS Generation 1 Human Interleukin-18 Decoy-Resistant Variants Amino AcidSequences

hCS1 YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISTYKDSQPRGKAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRDVPGHKHKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 34) hCS2YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISTYKDKQPRAKAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRDVPGHKHKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTIQNED (SEQ ID NO: 35) hCS3RFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISTYKDSQPRGKAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRDVPGHKHKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 36) hCS4RFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISTYRDSQPRGKAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRNVPGHKYKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 37) hC4YFGKLESQLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISTYKDKQPRTKAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFORRVPGHHNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQKED (SEQ ID NO: 38) hA8YFGKLESRLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISKYKDKQPRAQAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRDVPGHKHKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTIQNED (SEQ ID NO: 39) hD6YFGKLESRLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISDYKDKQPRAXAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRDVPGHKHKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTIQNED (SEQ ID NO: 40) hH12YFGKHESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISTYRDSQPRGKAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRDVPGHNNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTTQNED (SEQ ID NO: 41) hB11YFGKIESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISKYKDKQPRAQAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRKVPGHQHKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQKED (SEQ ID NO: 42) hC3YFGKIESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISTYKDRQPRGKAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFERDVPGHHHKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIM FTIQNED (SEQ ID NO: 43)hC2 YFGKIESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISTYKDKQPRGKAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRDVPGHKHKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIM FTTQHED (SEQ ID NO: 44)hG10 YFGKIESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISTYKDKQPRAKAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRRVPGHHHKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIM FTIQKED (SEQ ID NO: 45)hG1 YFGKIESRLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISTYKDKQPRGKAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRDVPGHDYKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIM FTIQKED (SEQ ID NO: 46)hF1 YFGKYESRLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISTYRDSQPRGKAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRDVPGHEHKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQKED (SEQ ID NO: 47) hD2HFGKYESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISTYRDSQPRGKAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRDVPGHHNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQKED (SEQ ID NO: 48) hA1RFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISTYRDSQPRAKAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRDVPGHQHKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTAQKED (SEQ ID NO: 49) hB3RFGKLESRLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISDYRDSQPRGRAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFKRNVPGHKYKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQHED (SEQ ID NO: 50) hB4RFGKLESRLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISNYRDSQPRGQAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFKRRVPGHNHKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQKED (SEQ ID NO: 51) hH3RFGKLESRLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISTYKDSQPRGKAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRDVPGHKHKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 52) hH5RFGKHESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISTYRDSQPRGKAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFERNVPGHKYKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 53) hH4RFGKLESRLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISTYRDSQPRAKAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFERDVPGHQHKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTIQXED (SEQ ID NO: 54) hE1RFGKLESRLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISTYRDSQPRTKAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRNVPGHHDKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQHED (SEQ ID NO: 55) hG2RFGKLESRLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISTYKDSQPRAKAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFERDVPGHQHKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTIQKED (SEQ ID NO: 56) hB9RFGKHESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISTYRDSQPRGKAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFERNVPGHKYKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 57) hE12RFGKYESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISTYKDSQPRTKAVTISVKCEKISTLSCDNKIISFKEMNPPDNIKDTKSDIIFFQRDVPGHKHKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 58) hC5RFGKLESRLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISTYRDSQPRTKAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRKVPGHNHKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQKED (SEQ ID NO: 59)Generation 2 Human Interleukin-18 Decoy-Resistant Variants Amino AcidSequences

5-18 YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISEYKDSELRGRAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFPRAVPGHNRKVQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 73) 5-29YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISKYKDSAGRGLAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFERDVPGHSNKVQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 74) 5-8YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISKYGDSAARGLAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHKRKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 75) 5-6YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISKYGDSRGRGLAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFERDVPGHNSKRQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 76) 5-27YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISKYGDSVPRGLAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFARAVPGHSRKTQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 77) 5-20YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISKYSDSGARGLAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFARAVPGHGRKTQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 78) 5-2YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISKYSDSKARGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFARDVPGHSSKRQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 79) 5-9YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISKYSDSLARGLAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRDVPGHSRKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 80) 5-42YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISKYSDSRARGLAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRNVPGHGRKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 81) 5-13YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISKYSDSRARGLAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFARSVPGHGRKTQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 82) 5-12YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISKYSDSRARGLAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFARDVPGHSGKRQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 83) 5-1YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISKYTDSRPRGLAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFERDVPGHSSKKQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 84) 5-33YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISKYTDSRARGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFERDVPGHNDKRQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 85) 5-21YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISRYKDSGKRGLAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFRRSVPGHSRKVQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 86) 6-31YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISKYGDSGARGLAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFERDVPGHSGKVQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 87) 6-20YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISKYGDSRPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRAVPGHNRKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 88) 6-12YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISKYSDSLARGLAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRDVPGHSRKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 89) 6-27YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISKYSDSRARGLAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFARSVPGHGRKTQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 90) 6-29YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISKYSDSRARGLAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRNVPGHGRKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 91) 5-26YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISKYGDSVPRGLAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFARAVPGHSRKTQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 191) 5-17YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISKYSDSRARGLAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFARSVPGHGRKTQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 192) 5-41YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISKYSDSRARGLAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFARDVPGHSGKRQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 193)Mouse Interleukin-18 Decoy-Resistant Variants Amino Acid Sequences

mCS1 NFGRLHCTTAVIRNINDQVLFVDKRQPVFEDMTDIDQSASEPQTRLIIYGYADSRVRGKAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFQKRVPGHNKMEFESSLYEGHFLACQKEDDAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 60) mCS2HFGRLHCTTAVIRNINDQVLFVDKRQPVFEDMTDIDQSASEPQTRLIIYAYGDSRARGKAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFQKRVPGHNKMEFESSLYEGHFLACQKEDDAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 61) mC1NFGRLHCTTAVIRNINDQVLFVDKRQPVFEDMTDIDQSASEPQTRLIIYAYVDRRLRGKAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFQKKVPGHNKMEFESSLYEGHFLACQKEDDAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 62) mA12NFGRLHCTTAVIRNINDQVLFVDKRQPVFEDMTDIDQSASEPQTRLIIYSYSDKHMRGKAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFQKLVPGHNKMEFESSLYEGHFLACQKEDDAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 63) mE8NFGRLHCTTAVIRNINDQVLFVDKRQPVFEDMTDIDQSASEPQTRLIIYVYTDGRRRGKAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFQKKVPGHDKMEFESSLYEGHFLACQKEDDAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 64) mC10HFGRLHCTTAVIRNINDQVLFVDKRQPVFEDMTDIDQSASEPQTRLIIYAYGDSHMRGKAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFQKQVPGHNKMEFESSLYEGHFLACQKEDDAFKLILKKKDENGDKSVMFTVTNLHQS (SEQ ID NO: 65) mB7HFGRLHCTTAVIRNINDQVLFVDKRQPVFEDMTDIDQSASEPQTRLIIYAYGDSNAGGRAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFQKKVPGHNKMEFESSLYEGHFLACQKEDDAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 66) mB1HFGRLHCTTAVIRNINDQVLFVDKRQPVFEDMTDIDQSASEPQTRLIIYGYADSDARAKAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFQKSVPGHNKMEFESSLYEGHFLACQKEDDAFKLILKKKDENGDKSVMFTVTNLHQS (SEQ ID NO: 67) mD1HFGRLHCTTAVIRNINDQVLFVDKRQPVFEDMTDIDQSASEPQTRLIIYGYSDRGSKGKAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFQKQVPGHNKMEFESSLYEGHFLACQKEDDAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 68) mH7YFGRLHCTTAVIRNINDQVLFVDKRQPVFEDMTDIDQSASEPQTRLIIYMYADRRARGKAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFQKKVPGHDKMEFESSLYEGHFLACQKEDDAFKLILKKKDENGDKSVMFTVTNLHQS (SEQ ID NO: 69) mA7YFGRLHCTTAVIRNINDQVLFVDKRQPVFEDMTDIDQSASEPQTRLIIYAYGDNRVRGKAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFQKRVPGHNKMEFESSLYEGHFLACQKEDDAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 70) mE1YFGRLHCTTAVIRNINDQVLFVDKRQPVFEDMTDIDQSASEPQTRLIIYGYGDSERGGRAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFQKRVPGHDKMEFESSLYEGHFLACQKEDDAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 71) mH3YFGRLHCTTAVIRNINDQVLFVDKRQPVFEDMTDIDQSASEPQTRLIIYTRTDGGQKGVAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFQKRVPGHDKMEFESSLYEGHFLACQKEDDAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 72)Human Decoy-to-the-Decoy (D2D) Variants Amino Acid Sequences

hD2D-5F12 HFGKLESKLSVIRNLNGQVLFIDQGNRPLFKDMTASDCRANAPRTIFIISFYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFIRSVPGADNKFQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 92) hD2D-5F11DFGKLESKLSVIRNLNDQVLFIDQGNRPLFADMTDNPCRSNAPRTIFIISFYKDSQPRGIAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFLRSVPGPDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 93) hD2D-5F10HFGKLESKLSVIRNLNGQVLFIDQGNRPLFADMEASPCRDNAPRTIFIISFYKDSQPRGLAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFLRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 94) hD2D-5F08LFGKLESKLSVIRNLNGQVLFIDQGNRPLFADMTSSPCRSRAPRTIFIISFYKDSQPRGFAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFIRSVPGHDNKIQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 95) hD2D-5F06HFGKLESKLSVIRNLNDQVLFIDQGNRPLFTDMESKPCRDSAPRTIFIISMYKDSQPRGIAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFIRSVPGHDNKFQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 96) hD2D-5F04YFGKLESKLSVIRNLNRQVLFIDQGNRPLFTDMTYKDCRDNAPRTIFIISFYKDSQPRGFAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFIRSVPGADNKIQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 97) hD2D-5F02HFGKLESKLSVIRNLNGQVLFIDQGNRPLFGDMEASPCRDNAPRTIFIISFYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFIRSVPGADNKLQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 98) hD2D-5F01HFGKLESKLSVIRNLNGQVLFIDQGNRPLFTDMTSSDCRDKAPRTIFIISFYKDSQPRGLAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFLRSVPGPDNKFQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 99) hD2D-5E10HFGKLESKLSVIRNLNGQVLFIDQGNRPLFADMESNRCRDSAPRTIFIISMYKDSQPRGFAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFLRSVPGHDNKIQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 100) hD2D-5E08YFGKLESKLSVIRNLNGQVLFIDQGNRPLFTDMTASPCRDNAPRTIFIISFYKDSQPRGLAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFLRSVPGHDNKIQFESSSYEGYFLACEKERSLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 101) hD2D-5E03DFGKLESKLSVIRNLNDQVLFIDQGNRPLFADMKSNVCRANAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFIRSVPGPDNKLQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 102) hD2D-5E02HFGKLESKLSVIRNLNGQVLFIDQGNRPLFGDMEASPCRAKAPRTIFIISIYKDSQPRGFAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFLRSVPGHDNKFQFESSSYEGYFLACEKERSLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 103) hD2D-5D10HFGKLESKLSVIRNLNGQVLFIDQGNRPLFADMASNRCRANAPRTIFIISMYKDSQPRGFAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFIRSVPGPDNKFQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 104) hD2D-5D08YFGKLESKLSVIRNLNDQVLFIDQGNRPLFADMKAKACRSNAPRTIFIISFYKDSQPRGFAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFLRSVPGADNKIQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 105) hD2D-5D06HFGKLESKLSVIRNLNHQVLFIDQGNRPLFTDMADNACRDNAPRTIFIISFYKDSQPRGLAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFIRSVPGDDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 106) hD2D-5D05YFGKLESKLSVIRNLNGQVLFIDQGNRPLFTDMKSNLCRSNAPRTIFIISFYKDSQPRGIAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFIRSVPGDDNKIQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 107) hD2D-5D03HFGKLESKLSVIRNLNGQVLFIDQGNRPLFRDMAASHCRDSAPRTIFIISIYKDSQPRGFAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFLRSVPGHDNKIQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 108) hD2D-5D02YFGKLESKLSVIRNLNDQVLFIDQGNRPLFADMASNPCRYKAPRTIFIISMYKDSQPRGLAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFLRSVPGADNKLQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 109) hD2D-5C10HFGKLESKLSVIRNLNGQVLFIDQGNRPLFTDMASNHCRYNAPRTIFIISMYKDSQPRGLAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFLRSVPGADNKIQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 110) hD2D-5C09HFGKLESKLSVIRNLNGQVLFIDQGNRPLFADMTDNPCRSRAPRTIFIISFYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFIRSVPGHDNKFQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 111) hD2D-5C08YFGKLESKLSVIRNLNGQVLFIDQGNRPLFTDMTASHCRSSAPRTIFIISLYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFLRSVPGHDNKFQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 112) hD2D-5C05YFGKLESKLSVIRNLNGQVLFIDQGNRPLFTDMEYRLCRANAPRTIFIISFYKDSHPRGLAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFLRSVPGDDNKLQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 113) hD2D-5C04YFGKLESKLSVIRNLNGQVLFIDQGNRPLFTDMESSLCRDNAPRTIFIISLYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFLRSVPGADNKFQFESSSYEGYFLACEKERSLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 114) hD2D-5C03YFGKLESKLSVIRNLNGQVLFIDQGNRPLFKDMEANDCRSSAPRTIFIISIYKDSQPRGLAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFIRSVPGADNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 115) hD2D-5B11DFGKLESKLSVIRNLNDQVLFIDQGNRPLFADMKASACRANAPRTIFIISMYKDSQPRGLAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFLRSVPGHDNKFQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 116) hD2D-5B10YFGKLESKLSVIRNLNGQVLFIDQGNRPLFGDMTAKHCRARAPRTIFIISFYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFIRSVPGADNKFQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 117) hD2D-5B06FFGKFESKLSVIRNLNGQVLFIDQGNRPLFTDMESKDCRDRAPRTIFIISFYKDSQPRGLAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFLRSVPGHDNKLQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 118) hD2D-5B05FFGKLESKLSVIRNLNGQVLFIDQGNRPLFADMASNHCRANAPRTIFIISLYKDSQPRGLAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFIRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 119) hD2D-5B02YFGKLESKLSVIRNLNGQVLFIDQGNRPLFADMTSKRCRDNAPRTIFIISLYKDSQPRGFAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFIRSVPGHDNKIQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 120) hD2D-5A09LFGKHESKLSVIRNLNGQVLFIDQGNRPLFGDMESSPCRYNAPRTIFIISFYKDSQPRGLAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFIRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 121) hD2D-5A02YFGKLESKLSVIRNLNAQVLFIDQGNRPLFTDMTASPCRSSAPRTIFIISLYKDSQPRGLAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFLRSVPGPDNKIQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 122) hD2D-CS1YFGKLESKLSVIRNLNGQVLFIDQGNRPLFADMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFLRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 123) hD2D-CS2YFGKLESKLSVIRNLNGQVLFIDQGNRPLFADMTSSDCRDNAPRTIFIISFYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFLRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 124) hD2D-CS3YFGKLESKLSVIRNLNGQVLFIDQGNRPLFADMESSDCRDNAPRTIFIISFYKDSQPRGLAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFLRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 125)Mouse Decoy-to-the-Decoy (D2D) Variants Amino Acid Sequences

mD2D-A5 YFGRYHCTTAVIRNINQQVLFVDKRQPVFADMGYTVQSASEPQTRLIIYMYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFLKEVPGHRKLEFESSLYEGHFLACQKEDEAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 126) mD2D-A6DFGRLHCTTAVIRNINDQVLFVDKRQPVFADMGSIAQSASEPQTRLIIYFYKDSEVRGLAVTLSVKDSKMYTLSCKNKIISFEEMDPPENIDDIQSDLIFFLKAVPGDNKIEFESSLYEGHFLACQKEATAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 127) mD2D-A7YFGRLHCTTAVIRNINGQVLFVDKRQPVFRDMADTVQSASEPQTRLIIYFYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFIKPVPGASKMEFESSLYEGHFLACQKEAGAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 128) mD2D-A8HFGRLHCTTAVIRNINDQVLFVDKRQPVFKDMEYTVQSASEPQTRLIIYMYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFIKAVPGDRKIEFESSLYEGHFLACQKEDNAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 129) mD2D-A9YFGRLHCTTAVIRNINAQVLFVDKRQPVFADMADKGQSASEPQTRLIIYMYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFLKPVPGDTKMEFESSLYEGHFLACQKEFGAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 130) mD2D-A11YFGRLHCTTAVIRNINEQVLFVDKRQPVFADMGDRHQSASEPQTRLIIYMYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFIKPVPGASKLEFESSLYEGHFLACQKEDDAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 131) mD2D-A12HFGRLHCTTAVIRNINDQVLFVDKRQPVFRDMGAIGQSASEPQTRLIIYFYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFIKPVPGDSKLEFESSLYEGHFLACQKEVDAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 132) mD2D-B4HFGRLHCTTAVIRNINSQVLFVDKRQPVFTDMGSIVQSASEPQTRLIIYMYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFIKGVPGDNKIEFESSLYEGHFLACQKEDRAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 133) mD2D-B7YFGRLHCTTAVIRNINSQVLFVDKRQPVFRDMEDTPQSASEPQTRLIIYFYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFIKRVPGDSKLEFESSLYEGHFLACQKEFEAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 134) mD2D-B11HFGRLHCTTAVIRNINAQVLFVDKRQPVFGDMTATVQSASEPQTRLIIYFYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFIKPVPGDSKLEFESSLYEGHFLACQKEDNAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 135) mD2D-B12NFGRLHCTTAVIRNINNQVLFVDKRQPVFKDMEYTLQSASEPQTRLIIYFYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFIKPVPGDNKLEFESSLYEGHFLACQKEYEAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 136) mD2D-C1YFGRLHCTTAVIRNINGQVLFVDKRQPVFADMEATRQSASEPQTRLIIYFYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFIKGVPGANKMEFESSLYEGHFLACQKEDGAFKLILKKKDENGDNSVMFTLTNLHQS (SEQ ID NO: 137) mD2D-C3NFGRLHCTTAVIRNINGQVLFVDKRQPVFADMRAILQSASEPQTRLIIYFYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFLKGVPGDNKLEFESSLYEGHFLACQKEDRAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 138) mD2D-C5YFGRLHCTTAVIRNINAQVLFVDKRQPVFADMEATAQSASEPQTRLIIYFYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFIKGVPGASKMEFESSLYEGHFLACQKEDGAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 139) mD2D-C6LFGRLHCTTAVIRNINGQVLFVDKRQPVFADMGATLQSASEPQTRLIIYMYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFLKPVPGDTKMEFESSLYEGHFLACQKEASAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 140) mD2D-C9NFGRLHCTTAVIRNINGQVLFVDKRQPVFEDMAYTVQSASEPQTRLIIYFYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFIKGVPGDSKMEFESSLYEGHFLACQKEYDAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 141) mD2D-C10DFGRLHCTTAVIRNINDQVLFVDKRQPVFKDMESKPQSASEPQTRLIIYFYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFLKAVPGASKLEFESSLYEGHFLACQKEANAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 142) mD2D-C11LFGRLHCTTAVIRNINGQVLFVDKRQPVFADMGDKVQSASEPQTRLIIYMYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFIKPVPGDNKLEFESSLYEGHFLACQKEDEAFKLILKTKDENGDKSVMFTLTNLHQS (SEQ ID NO: 143) mD2D-D1YFGRHHCTTAVIRNINQQVLFVDKRQPVFRDMAATRQSASEPQTRLIIYMYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFLKGVPGDNKMEFESSLYEGHFLACQKEDDAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 144) mD2D-D9NFGRLHCTTAVIRNINQQVLFVDKRQPVFTDMESIGQSASEPQTRLIIYFYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFLKAVPGANKLEFESSLYEGHFLACQKEDSAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 145) mD2D-D12FFGRHHCTTAVIRNINGQVLFVDKRQPVFGDMGDRVQSASEPQTRLIIYMYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFIKAVPGDSKIEFESSLYEGHFLACQKEDGAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 146) mD2D-E3VFGRHHCTTAVIRNINGQVLFVDKRQPVFKDMTYIDQSASEPQTRLIIYMYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFLKAVPGDTKMEFESSLYEGHFLACQKEAQAFKLILKKKDEIGDKSVMFTLTNLHQS (SEQ ID NO: 147) mD2D-E4NFGRLHCTTAVIRNINGQVLFVDKRQPVFADMTATRQSASEPQTRLIIYMYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFIKQVPGANKIEFESSLYEGHFLACQKEFRAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 148) mD2D-E5DFGRLHCTTAVIRNINGQVLFVDKRQPVFGDMAYIGQSASEPQTRLIIYFYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFIKAVPGHSKIEFESSLYEGHFLACQKESGAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 149) mD2D-E7YFGRLHCTTAVIRNINDQVLFVDKRQPVFRDMGSIAQSASEPQTRLIIYMYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFIKPVPGATKLEFESSLYEGHFLACQKEDGAFKLILKKKDENGDNSVMFTLTNLHQS (SEQ ID NO: 150) mD2D-E8YFGRLHCTTAVIRNINEQVLFVDKRQPVFTDMEAIGQSASEPQTRLIIYFYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFIKGVPGDRKMEFESSLYEGHFLACQKEDGAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 151) mD2D-E9FFGRLHCTTAVIRNINNQVLFVDKRQPVFEDMEYRLQSASEPQTRLIIYMYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFLKPVPGASKLEFESSLYEGHFLACQKESDAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 152) mD2D-E10NFGRLHCTTAVIRNINNQVLFVDKRQPVFADMEDRLQSASEPQTRLIIYMYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFLKGVPGDNKMEFESSLYEGHFLACQKEDHAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 153) mD2D-E11YFGRLHCTTAVIRNINAQVLFVDKRQPVFRDMGYILQSASEPQTRLIIYLYKDSEVRGLAVTLSVKESKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFLKPVPGDTKIEFESSLYEGHFLACQKEDNAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 154) mD2D-E12YFGRLHCTTAVIRNINDQVLFVDKRQPVFGDMADTAQSASEPQTRLIIYFYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFIKPVPGDSKMEFESSLYEGHFLACQKEADAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 155) mD2D-F3DFGRLHCTTAVIRNINGQVLFVDKRQPVFEDMAYIAQSASEPQTRLIIYFYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFIKPVPGDSKIEFESSLYEGHFLACQKEADAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 156) mD2D-F4NFGRLHCTTAVIRNINEQVLSVDKRQPVFRDMKYILQSASEPQTRLIIYFYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFLKGVPGDNKMEFESSLYEGHFLACQKEYGAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 157) mD2D-F5DFGRLHCTTAVIRNINEQVLFVDKRQPVFTDMAYILQSASEPQTRLIIYFYKDSEVRGLAVTLSVKESKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFIKAVPGDSKLEFESSLYEGHFLACQKEDTAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 158) mD2D-F7DFGRLHCTTAVIRNINNQVLFVDKRQPVFKDMESTAQSASEPQTRLIIYMYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFLKGVPGASKLEFESSLYEGHFLACQKEAGAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 159) mD2D-F8HFGRLHCTTAVIRNINEQVLFVDKRQPVFADMEAIGQSASEPQTRLIIYFYKDSEVRGLAVTLSVKESKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFIKGVPGDTKLEFESSLYAGHFLACQKEDGAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 160) mD2D-F9IFGRLHCTTAVIRNINEQVLFVDKRQPVFKDMRYIVQSASEPQTRLIIYFYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFIKEVPGASKLEFESSLYEGHFLACQKEDEAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 161) mD2D-G1YFGRLHCTTAVIRNINAQVLFVDKRQPVFTDMGYTLQSASEPQTRLIIYLYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFIKPVPGHNKIEFESSLYEGHFLACQKEDRAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 162) mD2D-G7NFGRLHCTTAVIRNINNQVLFVDKRQPVFRDMASTAQSASEPQTRLIIYMYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFIKGVPGANKIEFESSLYEGHFLACQKEDDAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 163) mD2D-G9DFGRLHCTTAVIRNINGQVLFVDKRQPVFEDMKDRAQSASEPQTRLIIYFYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFLKAVPGHSKMEFESSLYEGHFLACQKEDEAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 164) mD2D-H7NFGRLHCTTAVIRNINEQVLFVDKRQPVFADMTDIAQSASEPQTRLIIYMYKDSEVRGLAVTLSVKESKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFLKPVPGDIKMEFESSLYEGHFLACQKEYGAFKLILKKKDENGDNSVMFTLTNLHQS (SEQ ID NO: 165) mD2D-E1YFGRLHCTTAVIRNINEQVLFVDKRQPVFADMTDTLQSASEPQTRLIIYFYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFLKGVPGDNKMEFESSLYEGHFLACQKEDTAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 166) mD2D-G8YFGRLHCTTAVIRNINEQVLFVDKRQPVFADMTDTLQSASEPQTRLIIYFYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFLKGVPGDNKMEFESSLYEGHFLACQKEDTAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 167) mD2D-H3YFGRLHCTTAVIRNINEQVLFVDKRQPVFADMTDTLQSASEPQTRLIIYFYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFLKGVPGDNKMEFESSLYEGHFLACQKEDTAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 168) mD2D-A10HFGRLHCTTAVIRNINGQVLFVDKRQPVFKDMKYIVQSASEPQTRLIIYMYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFLKAVPGHSKIEFESSLYEGHFLACQKEDSAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 169) mD2D-H1HFGRLHCTTAVIRNINGQVLFVDKRQPVFKDMKYIVQSASEPQTRLIIYMYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFLKAVPGHSKIEFESSLYEGHFLACQKEDSAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 170) mD2D-F12YFGRLHCTTAVIRNINGQVLFVDKRQPVFEDMKAKAQSASEPQTRLIIYFYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFIKPVPGASKMEFESSLYEGHFLACQKEDGAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 171) mD2D-G10YFGRLHCTTAVIRNINGQVLFVDKRQPVFEDMKAKAQSASEPQTRLIIYFYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFIKPVPGASKMEFESSLYEGHFLACQKEDGAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 172) mD2D-G12YFGRLHCTTAVIRNINGQVLFVDKRQPVFEDMKAKAQSASEPQTRLIIYFYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFIKPVPGASKMEFESSLYEGHFLACQKEDGAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 173) mD2D-E2LFGRLHCTTAVIRNINGQVLFVDKRQPVFGDMGSIPQSASEPQTRLIIYFYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFIKHVPGATKMEFESSLYEGHFLACQKEDNAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 174) mD2D-G11LFGRLHCTTAVIRNINGQVLFVDKRQPVFGDMGSIPQSASEPQTRLIIYFYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFIKHVPGATKMEFESSLYEGHFLACQKEDNAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 175) mD2D-C4YFGRLHCTTAVIRNINSQVLFVDKRQPVFTDMAYTVQSASEPQTRLIIYFYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFIKAVPGDSKLEFESSLYEGHFLACQKEDNAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 176) mD2D-F11YFGRLHCTTAVIRNINSQVLFVDKRQPVFTDMAYTVQSASEPQTRLIIYFYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFIKAVPGDSKLEFESSLYEGHFLACQKEDNAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 177) mD2D-C2YFGRLHCTTAVIRNINGQVLFVDKRQPVFTDMGARVQSASEPQTRLIIYFYKDSEVRGLAVTLSVKDSKMYTLSCKNKIISFEEMDPPENIDDIQSDLIFFLKPVPGDNKLEFESSLYEGHFLACQKESGAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 178) mD2D-F10YFGRLHCTTAVIRNINGQVLFVDKRQPVFTDMGARVQSASEPQTRLIIYFYKDSEVRGLAVTLSVKDSKMYTLSCKNKIISFEEMDPPENIDDIQSDLIFFLKPVPGDNKLEFESSLYEGHFLACQKESGAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 179) mD2D-A2DFGRLHCTTAVIRNINGQVLFVDKRQPVFGDMKATGQSASEPQTRLIIYFYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFIKAVPGANKLEFESSLYEGHFLACQKEAGAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 180) mD2D-F6DFGRLHCTTAVIRNINGQVLFVDKRQPVFGDMKATGQSASEPQTRLIIYFYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFIKAVPGANKLEFESSLYEGHFLACQKEAGAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 181) mD2D-A1DFGRLHCTTAVIRNINSQVLFVDKRQPVFRDMGSIHQSASEPQTRLIIYFYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFLKAVPGANKLEFESSLYEGHFLACQKEDGAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 182) mD2D-E6DFGRLHCTTAVIRNINSQVLFVDKRQPVFRDMGSIHQSASEPQTRLIIYFYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFLKAVPGANKLEFESSLYEGHFLACQKEDGAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 183) mD2D-D4YFGRLHCTTAVIRNINEQVLFVDKRQPVFKDMKDKLQSASEPQTRLIIYFYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFLKGVPGDNKLEFESSLYEGHFLACQKEFGAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 184) mD2D-D6YFGRLHCTTAVIRNINEQVLFVDKRQPVFKDMKDKLQSASEPQTRLIIYFYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFLKGVPGDNKLEFESSLYEGHFLACQKEFGAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 185) mD2D-A3YFGRLHCTTAVIRNINGQVLFVDKRQPVFADMASTHQSASEPQTRLIIYFYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFLKGVPGANKIEFESSLYEGHFLACQKEDDAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 186) mD2D-A4YFGRLHCTTAVIRNINGQVLFVDKRQPVFADMASTHQSASEPQTRLIIYFYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFLKGVPGANKIEFESSLYEGHFLACQKEDDAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 187) mD2D-B10YFGRLHCTTAVIRNINGQVLFVDKRQPVFADMASTHQSASEPQTRLIIYFYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFLKGVPGANKIEFESSLYEGHFLACQKEDDAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 188) mD2D-B8YFGRLHCTTAVIRNINSQVLFVDKRQPVFGDMKYIVQSASEPQTRLIIYFYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFLKGVPGDTKMEFESSLYEGHFLACQKESGAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 189) mD2D-B9YFGRLHCTTAVIRNINSQVLFVDKRQPVFGDMKYIVQSASEPQTRLIIYFYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFLKGVPGDTKMEFESSLYEGHFLACQKESGAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 190)

SEQ ID NO: 89 (also referred to herein as “6-12”) (SEQ ID NO: 89)YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISKYSDSLARGLAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRDVPGHSRKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED SEQ ID NO: 6variant of SEQ ID NO: 89 (C38S/C68S) (SEQ ID NO: 6)YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSD S RDNAPRTIFIISKYSDSLARG LAVTISVK SEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRDVPGHSRKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED SEQ ID NO: 16variant of SEQ ID NO: 89 (C38S/C68G) (SEQ ID NO: 16)YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSD S RDNAPRTIFIISKYSDSLARG LAVTISVK GEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRDVPGHSRKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED SEQ ID NO: 17variant of SEQ ID NO: 89 (C38S/C68A) (SEQ ID NO: 17)YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSD S RDNAPRTIFIISKYSDSLARG LAVTISVK AEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRDVPGHSRKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED SEQ ID NO: 18variant of SEQ ID NO: 89 (C38S/C68V) (SEQ ID NO: 18)YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSD S RDNAPRTIFIISKYSDSLARG LAVTISVK VEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRDVPGHSRKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED SEQ ID NO: 19variant of SEQ ID NO: 89 (C38S/C68D) (SEQ ID NO: 19)YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSD S RDNAPRTIFIISKYSDSLARG LAVTISVK DEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRDVPGHSRKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED SEQ ID NO: 20variant of SEQ ID NO: 89 (C38S/C68E) (SEQ ID NO: 20)YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSD S RDNAPRTIFIISKYSDSLARG LAVTISVK EEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRDVPGHSRKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED SEQ ID NO: 21variant of SEQ ID NO: 89 (C38S/C68N) (SEQ ID NO: 21)YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSD S RDNAPRTIFIISKYSDSLARG LAVTISVK NEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRDVPGHSRKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED SEQ ID NO: 1 (SEQ ID NO: 1)YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSD S RDNAPRTIFIISKYSDSLARG LAVTISVK SEKISTLS S ENKIISFKEMNPPDNIKDTKSDIIFFQRDVPGHSRKMQFESSSY EGYFLA SEKERDLFKLILKKEDELGDRSIMFTVQNED SEQ ID NO: 2 (SEQ ID NO: 2)YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSD C RDNAPRTIFIISKYSDSLARG LAVTISVK SEKISTLS S ENKIISFKEMNPPDNIKDTKSDIIFFQRDVPGHSRKMQFESSSY EGYFLA SEKERDLFKLILKKEDELGDRSIMFTVQNED SEQ ID NO: 3 (SEQ ID NO: 3)YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSD S RDNAPRTIFIISKYSDSLARG LAVTISVK CEKISTLS S ENKIISFKEMNPPDNIKDTKSDIIFFQRDVPGHSRKMQFESSS YEGYFLA SEKERDLFKLILKKEDELGDRSIMFTVQNED SEQ ID NO: 4 (SEQ ID NO: 4)YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSD S RDNAPRTIFIISKYSDSLARG LAVTISVK SEKISTLS C ENKIISFKEMNPPDNIKDTKSDIIFFQRDVPGHSRKMQFESSS YEGYFLA SEKERDLFKLILKKEDELGDRSIMFTVQNED SEQ ID NO: 5 (SEQ ID NO: 5)YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSD S RDNAPRTIFIISKYSDSLARG LAVTISVK SEKISTLS S ENKIISFKEMNPPDNIKDTKSDIIFFQRDVPGHSRKMQFESSSY EGYFLA CEKERDLFKLILKKEDELGDRSIMFTVQNED SEQ ID NO: 7 (SEQ ID NO: 7)YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSD S RDNAPRTIFIISKYSDSLARG LAVTISVK CEKISTLS S ENKIISFKEMNPPDNIKDTKSDIIFFQRDVPGHSRKMQFESSS YEGYFLA CEKERDLFKLILKKEDELGDRSIMFTVQNED SEQ ID NO: 8 (SEQ ID NO: 8)YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSD S RDNAPRTIFIISKYSDSLARG LAVTISVK CEKISTLS C ENKIISFKEMNPPDNIKDTKSDIIFFQRDVPGHSRKMQFESSS YEGYFLA SEKERDLFKLILKKEDELGDRSIMFTVQNED SEQ ID NO: 9 (SEQ ID NO: 9)YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSD C RDNAPRTIFIISKYSDSLARG LAVTISVK SEKISTLS S ENKIISFKEMNPPDNIKDTKSDIIFFQRDVPGHSRKMQFESSSY EGYFLA CEKERDLFKLILKKEDELGDRSIMFTVQNED SEQ ID NO: 10 (SEQ ID NO: 10)YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSD C RDNAPRTIFIISKYSDSLARG LAVTISVK SEKISTLS C ENKIISFKEMNPPDNIKDTKSDIIFFQRDVPGHSRKMQFESSS YEGYFLA SEKERDLFKLILKKEDELGDRSIMFTVQNED SEQ ID NO: 11 (SEQ ID NO: 11)YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSD C RDNAPRTIFIISKYSDSLARG LAVTISVK CEKISTLS S ENKIISFKEMNPPDNIKDTKSDIIFFQRDVPGHSRKMQFESSS YEGYFLA SEKERDLFKLILKKEDELGDRSIMFTVQNED SEQ ID NO: 12 (SEQ ID NO: 12)YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSD S RDNAPRTIFIISKYSDSLARG LAVTISVK CEKISTLS C ENKIISFKEMNPPDNIKDTKSDIIFFQRDVPGHSRKMQFESSS YEGYFLA CEKERDLFKLILKKEDELGDRSIMFTVQNED SEQ ID NO: 13 (SEQ ID NO: 13)YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSD C RDNAPRTIFIISKYSDSLARG LAVTISVK SEKISTLS C ENKIISFKEMNPPDNIKDTKSDIIFFQRDVPGHSRKMQFESSS YEGYFLA CEKERDLFKLILKKEDELGDRSIMFTVQNED SEQ ID NO: 14 (SEQ ID NO: 14)YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSD C RDNAPRTIFIISKYSDSLARG LAVTISVK CEKISTLS S ENKIISFKEMNPPDNIKDTKSDIIFFQRDVPGHSRKMQFESSS YEGYFLA CEKERDLFKLILKKEDELGDRSIMFTVQNED SEQ ID NO: 15 (SEQ ID NO: 15)YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSD C RDNAPRTIFIISKYSDSLARG LAVTISVK CEKISTLS C ENKIISFKEMNPPDNIKDTKSDIIFFQRDVPGHSRKMQFESSS YEGYFLA SEKERDLFKLILKKEDELGDRSIMFTVQNED Example SUMO tag (SEQ ID NO: 26)DSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQAPEDLDMEDNDIIEAHREQIGG Example His tagged SUMO tag(SEQ ID NO: 27)

DSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQAPEDLDMEDNDIIEAHREQIGGExample SUMO protease (SEQ ID NO: 28)LVPELNEKDDDQVQKALASRENTQLMNRDNIEITVRDFKTLAPRRWLNDTIIEFFMKYIEKSTPNTVAFNSFFYTNLSERGYQGVRRWMKRKKTQIDKLDKIFTPINLNQSHWALGIIDLKKKTIGYVDSLSNGPNAMSFAILTDLQKYVMEESKHTIGEDFDLIHLDCPQQPNGYDCGIYVCMNTLYGSADAPLDFDYKDAIRMRRFIAHLILTDALKExample His tagged SUMO protease (SEQ ID NO: 29)

LVPELNEKDDDQVQKALASRENTQLMNRDNIEITVRDFKTLAPRRWLNDTHIEFFMKYIEKSTPNTVAFNSFFYTNLSERGYQGVRRWMKRKKTQIDKLDKIFTPINLNQSHWALGIIDLKKKTIGYVDSLSNGPNAMSFAILTDLQKYVMEESKHTIGEDFDLIHLDCPQQPNGYDCGIYVCMNTLYGSADAPLDFDYKDAIRMRRFIAHLILT DALKExemplary Non-Limiting Aspects of the Disclosure

Aspects, including embodiments, of the present subject matter describedabove may be beneficial alone or in combination, with one or more otheraspects or embodiments. Without limiting the foregoing description,certain non-limiting aspects of the disclosure are provided below in SETA and SET B. As will be apparent to those of ordinary skill in the artupon reading this disclosure, each of the individually numbered aspectsmay be used or combined with any of the preceding or followingindividually numbered aspects. This is intended to provide support forall such combinations of aspects and is not limited to combinations ofaspects explicitly provided below. It will be apparent to one ofordinary skill in the art that various changes and modifications can bemade without departing from the spirit or scope of the invention.

What is claimed is:
 1. A method of promoting interleukin-18 (IL-18)signaling activity in a human subject in need thereof, the methodcomprising administering an effective amount of a human decoy-resistant(DR) modified IL-18 polypeptide to the human subject in need thereof,the human DR modified IL-18 polypeptide comprising: (i) one or moresubstitution mutations selected from the group consisting of: (1)Tyrosine-1 to histidine, or Tyrosine-1 to arginine, (2) Leucine-5 tohistidine, Leucine-5 to isoleucine, or Leucine-5 to tyrosine, (3)Lysine-8 to glutamine, or Lysine-8 to arginine, (4) Methionine-51 tothreonine, Methionine-51 to lysine, Methionine-51 to aspartic acid,Methionine-51 to asparagine, Methionine-51 to glutamic acid, orMethionine-51 to arginine, (5) Lysine-53 to arginine, Lysine 53-glycine,Lysine-53 to serine, or Lysine-53 to threonine, (6) Serine-55 to lysine,or Serine-55 to arginine, (7) Glutamine-56 to glutamic acid,Glutamine-56 to alanine, Glutamine-56 to arginine, Glutamine-56 tovaline, Glutamine-56 to glycine, Glutamine-56 to lysine, or Glutamine-56to leucine, (8) Proline-57 to leucine, Proline-57 to glycine, Proline-57to alanine, or Proline-57 to lysine, (9) Glycine-59 to threonine, orGlycine-59 to alanine, (10) Methionine-60 to lysine, Methionine-60 toglutamine, Methionine-60 to arginine, or Methionine-60 to leucine, (11)Glutamic acid-77 to aspartic acid, (12) Glutamine-103 to glutamic acid,Glutamine-103 to lysine, Glutamine-103 to proline, Glutamine-103 toalanine, or Glutamine-103 to arginine, (13) Serine-105 to arginine,Serine-105 to aspartic acid, Serine-105 to lysine, Serine-105 toasparagine, or Serine-105 to alanine, (14) Aspartic acid-110 tohistidine, Aspartic acid-110 to lysine, Aspartic acid-110 to asparagine,Aspartic acid-110 to glutamine, Aspartic acid-110 to glutamic acid,Aspartic acid-110 to serine, or Aspartic acid-110 to glycine, (15)Asparagine-111 to histidine, Asparagine-111 to tyrosine, Asparagine-111to aspartic acid, Asparagine-111 to arginine, Asparagine-111 to serine,or Asparagine-111 to glycine, (16) Methionine-113 to valine,Methionine-113 to arginine, Methionine-113 to threonine, orMethionine-113 to lysine, (17) Valine-153 to isoleucine, Valine-153 tothreonine, or Valine-153 to alanine, and (18) Asparagine-155 to lysine,or Asparagine-155 to histidine, relative to wild-type (WT) IL-18 as setforth in SEQ ID NO: 30; and (ii) mutations at amino acid positionsCysteine-38 and Cysteine-68, relative to WT IL-18 as set forth in SEQ IDNO: 30, wherein the mutation at position Cysteine-38 is a substitutionof Cysteine-38 to serine and the mutation at Cysteine-68 is asubstitution selected from the group consisting of: Cysteine-68 toserine, Cysteine-68 to glycine, Cysteine-68 to alanine, Cysteine-68 tovaline, Cysteine-68 to aspartic acid, Cysteine-68 to glutamic acid, andCysteine-68 to asparagine, thereby promoting IL-18 signaling activity.2. The method of claim 1, wherein the mutation at position Cysteine-68is a substitution of Cysteine-68 to glycine.
 3. The method of claim 1,wherein the mutation at position Cysteine-68 is a substitution ofCysteine-68 to alanine.
 4. The method of claim 1, wherein the mutationat position Cysteine-68 is a substitution of Cysteine-68 to asparticacid.
 5. The method of claim 1, wherein the mutation at positionCysteine-68 is a substitution of Cysteine-68 to asparagine.
 6. Themethod of claim 1, wherein the human DR modified IL-18 polypeptidecomprises an amino acid sequence with no pegylated amino acids .
 7. Themethod of claim 1, wherein the human DR modified IL-18 polypeptidecomprises mutations in at least two of the following positions:Tyrosine-1, Leucine-5, Lysine-8, Methionine-51, Lysine-53, Serine-55,Glutamine-56, Proline-57, Glycine-59, Methionine-60, Glutamic acid-77,Glutamine-103, Serine-105, Aspartic acid-110, Asparagine-111,Methionine-113, Valine-153, and Asparagine-155, relative to SEQ ID NO:30.
 8. The method of claim 1, wherein the human DR modified IL-18polypeptide comprises mutations in at least two of the followingpositions: Methionine-51, Lysine-53, Serine-55, Glutamine-56,Proline-57, Methionine-60, Glutamine-103, Serine-105, Aspartic acid-110,Asparagine-111, and Methionine-113, relative WT IL-18 as set forth inSEQ ID NO:
 30. 9. The method of claim 1, wherein the human DR modifiedIL-18 polypeptide comprises mutations at positions Methionine-51,Lysine-53, Glutamine-56, Aspartic acid-110, and Asparagine-111, relativeto WT IL-18 as set forth in SEQ ID NO:
 30. 10. The method of claim 9,wherein the human DR modified IL-18 polypeptide further comprisesmutations at positions Proline-57 and Methionine-60, relative to WTIL-18 as set forth in SEQ ID NO:
 30. 11. The method of claim 10, whereinthe human DR modified IL-18 polypeptide further comprises a mutation atposition Serine-105, relative to WT IL-18 as set forth in SEQ ID NO: 30.12. The method of claim 1, wherein the human DR modified IL-18polypeptide has at least four substitutions selected from the groupconsisting of: (1) Methionine-51 to threonine, Methionine-51 to lysine,Methionine-51 to aspartic acid, Methionine-51 to asparagine,Methionine-51 to glutamic acid, or Methionine-51 to arginine, (2)Lysine-53 to arginine, Lysine-53 to glycine, Lysine-53 to serine, orLysine-53 to threonine, (3) Glutamine-56 to glutamic acid, Glutamine-56to alanine, Glutamine-56 to arginine, Glutamine-56 to valine,Glutamine-56 to glycine, Glutamine-56 to lysine, or Glutamine-56 toleucine, (4) Proline-57 to leucine, Proline-57 to glycine, Proline-57 toalanine, or Proline-57 to lysine, (5) Methionine-60 to lysine,Methionine-60 glutamine, Methionine-60 to arginine, or Methionine-60 toleucine, (6) Glutamine-103 to glutamic acid, Glutamine-103 to lysine,Glutamine-103 to proline, Glutamine-103 to alanine, or Glutamine-103 toarginine, (7) Serine-105 to arginine, Serine-105 to aspartic acid,Serine-105 to lysine, Serine-105 to asparagine, or Serine-105 toalanine, (8) Aspartic acid-110 to histidine, Aspartic acid-110 tolysine, Aspartic acid-110 to asparagine, Aspartic acid-110 to glutamine,Aspartic acid-110 to glutamic acid, Aspartic acid-110 to serine, orAspartic acid-110 to glycine, (9) Asparagine-111 to histidine,Asparagine-111 to tyrosine, Asparagine-111 to aspartic acid,Asparagine-111 to arginine, Asparagine-111 to serine, or Asparagine-111to glycine, and (10) Methionine-113 to valine, Methionine-113 toarginine, Methionine-113 to threonine, or Methionine-113 to lysine,relative to SEQ ID NO:
 30. 13. The method of claim 1, wherein the humanDR modified IL-18 polypeptide has at least five substitutions selectedfrom the group consisting of: (1) Methionine-51 to glutamic acid,Methionine-51 to arginine, Methionine-51 to lysine, Methionine-51 tothreonine, Methionine-51 to aspartic acid, or Methionine-51 toasparagine; (2) Lysine-53 to glycine, Lysine-53 to serine, Lysine-53 tothreonine, or Lysine-53 to arginine; (3) Glutamine-56 to glycine,Glutamine-56 to arginine, Glutamine-56 to leucine, Glutamine-56 toglutamic acid, Glutamine-56 to alanine, Glutamine-56 to valine, orGlutamine-56 to lysine; (4) Aspartic acid-110 to serine, Asparticacid-110 to asparagine, Aspartic acid-110 to glycine, Aspartic acid-110to lysine, Aspartic acid-110 to histidine, Aspartic acid-110 toglutamine, or Aspartic acid-110 to glutamic acid; (5) Asparagine-111 toglycine, Asparagine-111 to arginine, Asparagine-111 to serine,Asparagine-111 to aspartic acid, Asparagine-111 to histidine, orAsparagine-111 to tyrosine; (6) Proline-57 to alanine, Proline-57 toleucine, Proline-57 to glycine, or Proline-57 lysine; and (7)Methionine-60 to leucine, Methionine-60 to arginine, Methionine-60 tolysine, or Methionine-60 to glutamine, relative to SEQ ID NO:
 30. 14.The method of claim 13, wherein the human DR modified IL-18 polypeptidefurther comprises the substitution Serine-105 to aspartic acid,Serine-105 to alanine, Serine-105 to asparagine, Serine-105 to arginine,or Serine-105 to lysine relative to SEQ ID NO:
 30. 15. The method ofclaim 14, wherein the human DR modified IL-18 polypeptide comprisessubstitutions of Methionine-51 to lysine, Lysine-53 to serine,Glutamine-56 to leucine, Aspartic acid-110 to serine, and Asparagine-111to arginine, relative to SEQ ID NO:
 30. 16. The method of claim 15,wherein the human DR modified IL-18 polypeptide further comprisessubstitutions of Proline-57 to alanine, Methionine-60 to leucine, andSerine-105 to aspartic acid, relative to SEQ ID NO:
 30. 17. The methodof claim 16, wherein the mutations at amino acid position Cysteine-68,relative to WT IL-18 as set forth in SEQ ID NO: 30, is a Cysteine-68 toserine substitution.
 18. The method of claim 16, wherein the mutation atamino acid position Cysteine-68, relative to WT IL-18 as set forth inSEQ ID NO: 30, is a Cysteine-68 to glycine substitution.
 19. The methodof claim 16, wherein the mutation at amino acid position Cysteine-68,relative to WT IL-18 as set forth in SEQ ID NO: 30, is a Cysteine-68 toalanine substitution.
 20. The method of claim 16, wherein the mutationat amino acid position Cysteine-68, relative to WT IL-18 as set forth inSEQ ID NO: 30, is a Cysteine-68 to aspartic acid substitution.
 21. Themethod of claim 16, wherein the mutation at amino acid positionCysteine-68, relative to WT IL-18 as set forth in SEQ ID NO: 30, is aCysteine-68 to asparagine substitution.
 22. A composition comprising ahuman decoy-resistant (DR) modified IL-18 polypeptide, wherein the humanDR modified IL-18 specifically binds to IL-18 receptor (IL-18R) butexhibits reduced binding to IL-18 binding protein (IL-18BP) compared tothe wildtype (WT) IL-18, wherein the human DR modified IL-18 polypeptidecomprises: (i) one or more mutations selected from the group consistingof: (1) Tyrosine-1 to histidine, or Tyrosine-1 to arginine, (2)Leucine-5 to histidine, Leucine-5 to isoleucine, or Leucine-5 totyrosine, (3) Lysine-8 to glutamine, or Lysine-8 to arginine, (4)Methionine-51 to threonine, Methionine-51 to lysine, Methionine-51 toaspartic acid, Methionine-51 to asparagine, Methionine-51 to glutamicacid, or Methionine-51 to arginine, (5) Lysine-53 to arginine, Lysine53-glycine, Lysine-53 to serine, or Lysine-53 to threonine, (6)Serine-55 to lysine, or Serine-55 to arginine, (7) Glutamine-56 toglutamic acid, Glutamine-56 to alanine, Glutamine-56 to arginine,Glutamine-56 to valine, Glutamine-56 to glycine, Glutamine-56 to lysine,or Glutamine-56 to leucine, (8) Proline-57 to leucine, Proline-57 toglycine, Proline-57 to alanine, or Proline-57 to lysine, (9) Glycine-59to threonine, or Glycine-59 to alanine, (10) Methionine-60 to lysine,Methionine-60 to glutamine, Methionine-60 to arginine, or Methionine-60to leucine, (11) Glutamic acid-77 to aspartic acid, (12) Glutamine-103to glutamic acid, Glutamine-103 to lysine, Glutamine-103 to proline,Glutamine-103 to alanine, or Glutamine-103 to arginine, (13) Serine-105to arginine, Serine-105 to aspartic acid, Serine-105 to lysine,Serine-105 to asparagine, or Serine-105 to alanine, (14) Asparticacid-110 to histidine, Aspartic acid-110 to lysine, Aspartic acid-110 toasparagine, Aspartic acid-110 to glutamine, Aspartic acid-110 toglutamic acid, Aspartic acid-110 to serine, or Aspartic acid-110 toglycine, (15) Asparagine-111 to histidine, Asparagine-111 to tyrosine,Asparagine-111 to aspartic acid, Asparagine-111 to arginine,Asparagine-111 to serine, or Asparagine-111 to glycine, (16)Methionine-113 to valine, Methionine-113 to arginine, Methionine-113 tothreonine, or Methionine-113 to lysine, (17) Valine-153 to isoleucine,Valine-153 to threonine, or Valine-153 to alanine, and (18)Asparagine-155 to lysine, or Asparagine-155 to histidine, relative towild-type (WT) IL-18 as set forth in SEQ ID NO: 30; and (ii) mutationsat amino acid positions Cysteine-38 and Cysteine-68, relative towild-type (WT) IL-18 as set forth in SEQ ID NO: 30, wherein the mutationat position Cysteine-38 is a substitution of Cysteine-38 to serine andthe mutation at Cysteine-68 is a substitution selected from the groupconsisting of: Cysteine-68 to serine, Cysteine-68 to glycine,Cysteine-68 to alanine, Cysteine-68 to valine, Cysteine-68 to asparticacid, Cysteine-68 to glutamic acid, and Cysteine-68 to asparagine. 23.The composition of claim 22, wherein the one or more mutations comprisea substitution of Methionine-51 to threonine, Methionine-51 to lysine,Methionine-51 to aspartic acid, Methionine-51 to asparagine,Methionine-51 to glutamic acid, or Methionine-51 to arginine, relativeto WT IL-18 as set forth in SEQ ID NO:
 30. 24. The composition of claim22, wherein the one or more mutations comprise a substitution ofLysine-53 to glycine, Lysine-53 to serine, Lysine-53 to threonine, orLysine-53 to arginine, relative to WT IL-18 as set forth in SEQ ID NO:30.
 25. The composition of claim 22, wherein the one or more mutationscomprise a substitution of Glutamine-56 to glutamic acid, Glutamine-56to alanine, Glutamine-56 to arginine, Glutamine-56 to valine,Glutamine-56 to glycine, Glutamine-56 to lysine, or Glutamine-56 toleucine, relative to WT IL-18 as set forth in SEQ ID NO:
 30. 26. Thecomposition of claim 22, wherein the one or more mutations comprise asubstitution of Proline-57 to leucine, Proline-57 to glycine, Proline-57to alanine, or Proline-57 to lysine, relative to WT IL-18 as set forthin SEQ ID NO:
 30. 27. The composition of claim 22, wherein the one ormore mutations comprise a substitution of Aspartic acid-110 tohistidine, Aspartic acid-110 to lysine, Aspartic acid-110 to asparagine,Aspartic acid-110 to glutamine, Aspartic acid-110 to glutamic acid,Aspartic acid-110 to serine, Aspartic acid-110 to glycine, relative toWT IL-18 as set forth in SEQ ID NO:
 30. 28. The composition of claim 22,wherein the one or more mutations comprise a substitution ofAsparagine-111 to histidine, Asparagine-111 to tyrosine Asparagine-111to aspartic acid, Asparagine-111 to arginine, Asparagine-111 to serine,or Asparagine-111 to glycine, relative to WT IL-18 as set forth in SEQID NO:
 30. 29. The composition of claim 22, wherein the one or moremutations comprise a substitution of Serine-105 to aspartic acid,Serine-105 to alanine, Serine-105 to asparagine, Serine-105 to arginine,Serine 105 to aspartic acid, or Serine-105 to lysine, relative to WTIL-18 as set forth in SEQ ID NO:
 30. 30. The composition of claim 22,wherein the one or more mutations comprise a substitution ofMethionine-60 to lysine, Methionine-60 to glutamine, Methionine-60 toarginine, or Methionine-60 to leucine, relative to WT IL-18 as set forthin SEQ ID NO:
 30. 31. The composition of claim 22, wherein the mutationsat amino acid positions Cysteine-38 and Cysteine-68 comprisesubstitutions of Cysteine-38 to serine and Cysteine-68 to aspartic acid,relative to WT IL-18 as set forth in SEQ ID NO: 30.